WO2008011513A2 - Composés et méthodes de modulation de l'angiogenèse - Google Patents
Composés et méthodes de modulation de l'angiogenèse Download PDFInfo
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- WO2008011513A2 WO2008011513A2 PCT/US2007/073877 US2007073877W WO2008011513A2 WO 2008011513 A2 WO2008011513 A2 WO 2008011513A2 US 2007073877 W US2007073877 W US 2007073877W WO 2008011513 A2 WO2008011513 A2 WO 2008011513A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present invention relates to compounds and methods of modulating angiogenesis and particularly relates to compounds and methods of modulating tumor growth and angiogenesis.
- angiogenesis involves coordinated endothelial cell (EC) proliferation, invasion, migration, and tube formation. This process is known to be induced by vascular growth factors and its receptors in coordination with extracellular matrix interacting molecules such as integrins. Integrins are heterodimeric transmembrane receptors, which play central roles in cell adhesion, migration, proliferation, differentiation and programmed cell death. ⁇ v p 3 integrin is a major integrin expressed on proliferating endothelial cells during angiogenesis and vascular remodeling.
- O v ⁇ 3 integrin ligation either by blocking antibody (LM609 or Vitaxin) or by cyclic peptide antagonists (RGD) prevents blood vessel formation in mouse retina, rabbit cornea, chick chorioallantoic membrane, and human skin transplanted onto athymic mice.
- function-blocking anti- ⁇ j antibody that recognizes at least three integrins ( ⁇ ii b ⁇ 3j ⁇ v ⁇ 3 and CX M P 2 ) has been shown to be beneficial in high risk angioplasty patients in part due to the blockade of ⁇ v p 3 .
- the present invention relates to a method of modulating angiogenesis in a cell population.
- the method includes contacting the cell population that comprises cells expressing ⁇ v ⁇ 3 integrin and VEGFR2 with a therapeutically effective amount of an agent that stimulates or inhibits complexing of O v ⁇ 3 integrin and VEGFR2.
- the agent does not substantially inhibit natural or native ligand (e.g., vitronectin) binding to the ⁇ v ⁇ 3 integrin.
- the agent can inhibit tyrosine phosphorylation of the ⁇ v ⁇ 3 integrin and/or tyrosine phosphorylation of VEGFR2 upon VEGF stimulation.
- the agent can also compete with ⁇ v ⁇ 3 integrin for interaction with VEGFR2.
- the agent can inhibit tyrosine phosphorylation of a tyrosine of the P 3 subunit of the ⁇ v ⁇ 3 integrin.
- the agent can inhibit tyrosine phosphorylation of a tyrosine of the cytoplasmic tail of the ⁇ 3 subunit of the O v ⁇ 3 integrin, and more particularly, inhibit tyrosine phosphorylation of tyrosine 747 of the ⁇ 3 subunit of the ⁇ v ⁇ 3 integrin.
- the agent can comprise a peptide having a peptide sequence of about 5 to about 50 amino acids in length.
- the peptide can have a sequence that corresponds to a portion of the peptide sequence of at least one of ⁇ v ⁇ 3 integrin or VEGFR2.
- the portion of the peptide sequence of at least one of 0 ⁇ 3 integrin or VEGFR2 can contain a tyrosine residue, which is capable of being phosphorylated upon complex formation of the ⁇ v p 3 integrin and VEGFR2.
- the agent can comprise a peptide that has a sequence that corresponds to a portion of the peptide sequence of the cytoplasmic domain of the ⁇ 3 subunit of the Oy ⁇ 3 integrin.
- the portion of the peptide sequence of the cytoplasmic domain of the p3 subunit of the ctvp3 integrin can contain a tyrosine residue (e.g., tyrosine -747), which is capable of being phosphorylated upon complex formation of the ⁇ v ⁇ 3 integrin and VEGFR2.
- the peptide can comprise, for example, a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1, and SEQ ID NO: 12.
- the agent can comprise a c-SRC kinase inhibitor that inhibits tyrosine phosphorylation of 0 ⁇ 3 integrin.
- the c-SRC kinase inhibitor can include a 3-(4,5,6,7-tetrahydroinol-2-ylmethylidene)-2-indolinone derivative.
- Examples of a 3-(4,5,6,7-tetrahydroinol-2-ylmethylidene)-2-indolinone derivatives include 2-oxo-3 (4,5,6, 7-tetrahydro-l -H-indol-2-ylmethylene)-2,3-dihydro-lH- indole-5-sulfonic acid dimethylamide and 2-oxo-3(4,5,6, 7-tetrahydro-l-H-indol-2- ylmemylene)-2,3-dihydro-lH-indole-5-sulfbnic acid amide.
- the agent can comprise an antisense oligonucleotide, interfering nucleotide, or antibody that inhibits expression or activity of SRC-kinase.
- the present invention also relates to a method of inhibiting ang ⁇ ogenesis in a tissue.
- the method comprises administering to the tissue a therapeutically effective amount of an agent that inhibits complex formation of ct v ⁇ 3 integrin and VEGFR2.
- the agent does not inhibit natural ligand binding to the Ov ⁇ 3 integrin.
- the agent can inhibit tyrosine phosphorylation of the 0 ⁇ 3 integrin and/or tyrosine phosphorylation of VEGFR2 upon VEGF stimulation.
- the agent can also compete with O v p 3 integrin for interaction with VEGFR2.
- the agent can inhibit tyrosine phosphorylation of a tyrosine of the ⁇ 3 subunit of the ci v ⁇ 3 integrin.
- the agent can inhibit tyrosine phosphorylation of a tyrosine of the A-
- the agent can comprise a peptide having a peptide sequence of about 5 to about 50 amino acids in length.
- the peptide can have a sequence that corresponds to a portion of the peptide sequence of at least one of ⁇ v p 3 integral or VEGFR2.
- the portion of the peptide sequence of at least one of Ovp 3 integrin or VEGFR2 can contain a tyrosine residue, which is capable of being phosphorylated upon complex formation of the ⁇ v ⁇ 3 integrin and VEGFR2.
- the agent can comprise a peptide that has a sequence that corresponds to a portion of the peptide sequence of the cytoplasmic domain of the ⁇ 3 subunit of the ⁇ v ⁇ 3 integrin.
- the portion of the peptide sequence of the cytoplasmic domain of the ⁇ 3 subunit of the 0 ⁇ 3 integrin can contain a tyrosine residue (e.g., tyrosine -747), which is capable of being phosphorylated upon complex formation of the ⁇ v p3 integrin and VEGFR2.
- the peptide can comprise, for example, a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO:2 5 SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 5 and SEQ ID NO: 12.
- the agent can comprise a c-SRC kinase inhibitor that inhibits tyrosine phosphorylation of ⁇ v ⁇ 3 integrin.
- the c-SRC kinase inhibitor can include a 3-(4,5,6,7-tet ⁇ ahydroinol-2-ylmethylidene)-2-indolinone derivative.
- Examples of a 3-(4,5,6 J 7-tetrahydroinol-2-ylmethylidene)-2-indolinone derivatives include 2-oxo-3 (4,5 ,6, 7-tetrahydro- 1 -H-indol-2-ylmethylene)-2,3-dihydro- 1 H- indole-5-sulfonic acid dimethylamide and 2-oxo-3(4,5,6, 7-tetrahydro- 1-H-indol- 2- ylmethylene)-2,3-dihydro-lH-indole-5-sulfonic acid amide.
- the agent can comprise an antisense oligonucleotide, interfering nucleotide, or antibody that inhibits expression or activity of SRC-
- the present invention also relates to a method of treating an angiogenic disorder in a subject.
- the angiogenic disorder can include, for example, aberrant tumor growth and age-related macular degeneration.
- the method includes administering to cells expressing o v ⁇ 3 integrin and VEGFR2 of the subject a therapeutically effective amount of agent that inhibits complexing of the expressed ⁇ v ⁇ 3 integrin and VEGFR2.
- the agent does not inhibit natural ligand binding to the ⁇ y ⁇ 3 integrin.
- the agent can inhibit tyrosine phosphorylation of the ⁇ v ⁇ 3 integrin and/or tyrosine phosphorylation of VEGFR2 upon VEGF stimulation.
- the agent can also compete with ⁇ v ⁇ 3 integrin for interaction with VEGFR2.
- the agent can inhibit tyrosine phosphorylation of a tyrosine of the ⁇ 3 subunit of the ct v ⁇ 3 integrin.
- the agent can inhibit tyrosine phosphorylation of a tyrosine of the cytoplasmic tail of the ⁇ 3 subunit of the ⁇ Xv ⁇ 3 integrin, and more particularly, inhibit tyrosine phosphorylation of tyrosine 747 of the ⁇ 3 subunit of the ⁇ v ⁇ 3 integrin.
- the agent can comprise a peptide having a peptide sequence of about 5 to about 50 amino acids in length.
- the peptide can have a sequence that corresponds to a portion of the peptide sequence of at least one of Ovp3 integrin or VEGFR2.
- the portion of the peptide sequence of at least one of Ov ⁇ 3 integrin or VEGFR2 can contain a tyrosine residue, which is capable of being phosphorylated upon complex formation of the ⁇ v p 3 integrin and VEGFR2.
- the agent can comprise a peptide that has a sequence that corresponds to a portion of the peptide sequence of the cytoplasmic domain of the ⁇ 3 subunit of the ⁇ v ⁇ 3 integrin.
- the portion of the peptide sequence of the cytoplasmic domain of the ⁇ 3 subunit of the ⁇ 3 integrin can contain a tyrosine residue (e.g., tyrosine -747), which is capable of being phosphorylated upon complex formation of the Ct 1 ⁇ integrin and VEGFR2.
- the peptide can comprise, for example, a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.
- the agent can comprise a c-SRC kinase inhibitor that inhibits tyrosine phosphorylation of ⁇ v ⁇ 3 integrin.
- the c-SRC kinase inhibitor can include a 3-(4,5,6,7-tetrahydroinol-2-yL ⁇ iethylidene)-2-indolinone derivative.
- Examples of a 3-(4,5,6,7-tetrahydroinol-2-ylmethylidene)-2-indolinone derivatives include 2-oxo-3(4,5,6, 7-tetrahydro-l-H-indol-2-ylmethylene)-2,3-dihydro-lH- indole-5-sulfonic acid dimethylamide and 2-oxo-3(4,5,6, 7-tetrahydro-l-H-indol-2- ylmethylene)-2,3-dihydro-lH-indole-5 -sulfonic acid amide.
- the agent can comprise an antisense oligonucleotide, interfering nucleotide, or antibody that inhibits expression or activity of SRC
- the present invention further relates to a pharmaceutical composition that includes a synthetic peptide that modulates complex formation of ⁇ v p 3 integrin and VEGFR2 in tumor cells.
- the synthetic peptide can have a sequence that corresponds to a portion of the peptide sequence of at least one of ⁇ v ⁇ 3 integrin or VEGFR2.
- the portion of the peptide sequence of at least one of d v ⁇ 3 integrin or VEGFR2 can contain a tyrosine residue, which is capable of being phosphorylated upon complex formation of the ⁇ v ⁇ 3 integrin and VEGFR2.
- the synthetic peptide can include a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 5 and SEQ ID NO: 12.
- FIG. 1 illustrates extracellular matrix proteins and VEGF induce ⁇ integrin cytoplasmic tyrosine motif phosphorylation.
- A-B f ⁇ integrin tyrosine phosphorylation upon adhesion to integrin ligands.
- Wild type (A) and DiYF (B) cells were either held in suspension (lane-1) or plated on poly-L-lysine (lane-2), gelatin (lane-3,4) with (lane 4) or without (lane 3) pervanadate treatment, laminin (lane-5), collagen (lane-6), vitronectin (lane-7), fibronectin (lane-8), fibrinogen (lane-9) and incubated at 37 C for 60 mins.
- VEGF induces ⁇ 3 integrin tyrosine phosphorylation.
- WT and DiYF mouse lung EC were treated with 20 ng/ml of VEGF-A165 for 0 to 60 min as indicated. Phosphorylation status of ⁇ 3 Y 747 (upper panel) and P 3 Y 759 (middle panel) was assessed as described in A.
- FIG. 2 illustrates ⁇ 3 integrin cytoplasmic tyrosine motif is critical for endothelial cell adhesion, spreading and migration.
- A-B Adhesion and spreading of WT and DiYF endothelial cells on various integrin ligands. Plates were coated with 10 ⁇ g/ml vitronectin (VN), fibronectin (FN), lam ⁇ nin-1 (LM-I) 1 collagen (COLL) or bovine serum albumin (BSA) over night at 4 0 C.
- VN vitronectin
- FN fibronectin
- LM-I lam ⁇ nin-1
- BSA bovine serum albumin
- Wild type and DiYF mouse lung EC were harvested and re-suspended in serum free media at 5X10 5 cells/ml. 100 ⁇ l cell suspension was plated on each well coated with integrin ligands. After incubation at 37 C for 45 min wells were gently washed three times with DMEM and photographs were taken. The numbers of attached and spread cells per field were counted. Adhesion and spreading of WT EC on vitronectin was assigned a value of 100%. (P values were 0.000017 and 0.00003 for cell adhesion and spreading, respectively.) (C). Migration of WT and DiYF endothelial cells on various integrin ligands. Tissue culture inserts were coated with various integrin ligands.
- D VEGF-induced migration of WT and DiYF mouse lung EC. Migration assay was performed as described above using vitronectin as a substrate. The lower chamber contained 0-20 ng/ml of VEGF A 165. Cells were allowed to migrate, fixed, stained and photographed. Number of WT EC migrated in the absence of VEGF was referred as 100%.
- E-F Reduced migration of DiYF endothelial cells on ⁇ 3 integrin ligand.
- Wild type and DiYF EC were serum starved and wounded across the cell monolayer by scraping away a swath of cells.
- the data represents the results of three independent experiments. Images were acquired using a Leica DMIRB phase contrast microscope, objective 5X, and a Micromax RTE/CCD-1300-V-HS camera.
- Fig. 3 illustrates ⁇ 3 integrin tyrosine 747 and 759 mutations impair angiogenic properties of EC.
- A-B Functional ⁇ 3 integrin is essential for endothelial cell organization into precapillary cords.
- WT and DiYF mouse lung endothelial cells were collected and re-suspended in DMEM containing 10% FCS. Equal numbers of cells were seeded on Matrigel coated plates and cells were allowed to adhere. After 24 h cells were overlaid with Matrigel with or without 40 ng/ml VEGF and maintained in culture for 6-8 days. Three random fields were photographed periodically using phase contrast microscope (Leica) (panel A).
- FIG. 4 illustrates ⁇ 3 integrin cytoplasmic tyrosine mutations result in impaired capillary growth ex vivo.
- A-B ⁇ 3 integrin cytoplasmic tyrosine motif is required for normal microvessel growth. Wild type (upper panel) and DiYF (lower panel) mouse aortic rings were embedded in Matrigel and maintained at 37 C for six days. Microvessels outgrowths from aortic rings were observed periodically and photographed using Leica DMIRB phase contrast microscope. Numbers of microvessel sprouts from aortic rings were counted and represented as bar diagram (panel B).
- C-D Defective ⁇ 3 integrin tyrosine phosphorylation reduces VEGF-induced microvessel outgrowth.
- Wild type and DiYF mouse aortic rings were implanted in Matrigel with or without 40 ng/ml VEGF and maintained under aseptic condition for six days. Microvessel growths from implants were observed periodically and microphotographed. Representative images were shown in panel C. Numbers of microvessel outgrowth from aortic implants were counted and shown in panel D).
- E-G In ex vivo aortic ring assay using WT and DiYF aortic rings was performed in the presence of DMEM without any supplements (panel E), with endothelial growth supplement (panel F), or with 40 ng/ml VEGF together with endothelial growth supplement (panel G), Aortic rings were observed every two days, numbers of sprouts from each implant were counted and photographs were taken. Growth kinetics of wild type and DiYF aortic rings under various conditions were analyzed and represented as growth curves. The data represents the results of three independent experiments performed in triplicates. Images were acquired using a Leica DMIRB phase contrast microscope, objective 5X, and a Micromax RTE/CCD- 1300- V-HS camera.
- Fig. 5 illustrates ⁇ 3 integrin phosphorylation controls integrin activation.
- A-B DiYF EC exhibit reduced fibrinogen binding in response to growth factors. Wild type and DiYF mouse lung EC were serum starved for 4h and washed twice with IX PBS. These cells were incubated with FITC-fibrinogen in the presence or absence of 20 ng/ml VEGF for 45 min at 37 ° C. Cells were fixed with 0.4% formaldehyde, washed and analyzed by flow cytometry. Bars represent mean fluorescence intensity of three independent experiments performed in triplicates (panel A). Wild type and DiYF mouse lung endothelial cells were stimulated with ImM MnCl 2 .
- FIG. 6 illustrates ⁇ 3 integrin cytoplasmic tyrosine residues are required for VEGF induced VEGF receptor and ⁇ 3 integrin interaction.
- Wild type (C) and DiYF (D) mouse lung endothelial cells were either kept in suspension (lanes 1 and 2) or plated on vitronectin coated (lanes 3 and 4) or laminin coated (lanes 5 and 6) plates. These cells were treated with 20 ng/ml VEGF for 15 min (lanes 2, 4 and 6), cells were lysed, immunoprecipitated with rabbit-anti mouse p 3 integrin antibody and analyzed Western blot using rabbit-anti mouse VEGF receptor antibody.
- Fig. 7 illustrates knockdowns of beta subur ⁇ ts of integrins on endothelial cells by specific siRNAs.
- A-C HUVECs were transfected with control siRNA or integrin specific siRNA and cell lysates were analyzed for expression of ⁇ l (A), ⁇ 3 (B), or ⁇ 5 (C) integrin subunits using specific antibody. Densitometry analysis was performed and results are shown in bar graphs (lower panel).
- D-E Cell surface expression of ⁇ l (D), ⁇ 3 (E), or ⁇ 5 (F) integrin subunits in endothelial cells was assessed by FACS analysis.
- FIG. 8 illustrates specificity of integrins in reorganization on distinct ECM ligands.
- A-B HUVECs were transfected with control siRNA or siRNA specific for ⁇ l, ⁇ 3, or ⁇ 5 integrin. Wells of the microliter plates were coated with vitronectin, collagen, or laminin-1 and were incubated overnight at 4 0 C.
- siRNA-transfected EC were harvested and resuspended in serum-free media at 5 x 10 5 cells/mL.
- the cell suspension 100 ⁇ L was plated on a microtiter well coated with integrin ligand. After incubation at 37 0 C for 45 min, wells were gently washed three times with DMEM and photographs were taken (panel A). The numbers of attached cells per field were counted and untransfected cells adhered on the individual ECM ligand were assigned a value of 100% (panel B), Asterisks indicate significant difference over control (P ⁇ 0.0046). [0039] Fig.
- FIG. 9 illustrates Vitronectin ( ⁇ v p 3 ) and collagen ( ⁇ 5 ⁇ l) receptors regulate endothelial cell migration.
- A-B HUVECs were transfected with control siRNA or siRNA specific for ⁇ l, ⁇ 3 ? or ⁇ 5 integrin. These cells were grown to confluence on 12- well plates precoated with individual integrin ligand. Cells were serum starved and wounded across the cell monolayer by scraping away a swath of cells. Wells were rinsed twice with sterile PBS and further cultured in DMEM medium containing 2% FBS. Sites were photographed immediately after wounding (zero hour) and 12 h later using a phase contrast microscope (panel A).
- FIG. 10 illustrates ⁇ 3 integrin regulates endothelial cell morphogenesis in vitro.
- A-B HUVECs were transfected with control siRNA or siRNA specific for ⁇ l, ⁇ 3, or ⁇ 5 integrin. Cells were transferred to Matrigel coated plates and further incubated at 37 0 C for 8 h with or without 20 ng/mL VEGF. Endothelial capillary tubes formed in Matrigel were observed using an inverted phase contrast microscope and photographs were taken (panel A), Mean length of tubes from five random fields were measured using ImagePro software (panel B). Asterisks indicate significant difference over control.
- FIG. 11 illustrates activated ⁇ v ⁇ 3 integrin co-localizes with VEGFR-2 on endothelial cells.
- A To evaluate VEGF/VEGFR-2 dependent activation of Ov ⁇ 3 integrin, semiconfluent, serum starved HUVECs were induced with VEGF-A 165 or VEGFD ⁇ N ⁇ C. These cells were further incubated with WOW-I Fab fragment and goat anti-mouse IgG labeled with AlexaFluor 488. Fixed cells were then analyzed by flow cytometry.
- HUVECs were grown on the gelatin-coated glass coverslips.
- Fig. 12 illustrates the level of ⁇ v ⁇ 3 integrin activation is index for degree of tumor angiogenesis.
- A Activated Ov ⁇ 3 integrin co-localizes with VEGFR-2 on endothelial cells of proliferating blood vessel. Parallel prostate tumor tissue sections were cut and stained for WOW-I (activated ⁇ v ⁇ 3 integrin), CD31 (endothelial cell marker), and VEGFR-2. Blood vessels (revealed by CD31 staining) were positively stained for both WOW-I and VEGFR-2, indicating the co-localization of activated ctv ⁇ with VEGFR-2 in tumor vasculature.
- WOW-I activated ⁇ v ⁇ 3 integrin
- CD31 endothelial cell marker
- VEGFR-2 VEGFR-2
- FIG. 1 Frozen parallel prostate tumor sections were stained for activated ⁇ V ⁇ 3 integrin (WOW-I Fab) and VEGFR-2. Tissue sections were analyzed under a confocal microscope and photographs were taken.
- C-D Normal prostate tissue (panel Q and prostate tumor sections (panel D) were stained for CD-31 and WOW-I .
- Vascular density was increased at least by 6 times in prostate tumors compared to normal prostate tissue.
- E Vascular density was positively correlated with the density of WOW-I- positive vasculature in the two tissue samples. Asterisks indicate significant difference over normal tissue.
- Fig. 13 illustrates VEGF-induced VEGFR-2 phosphorylation is subordinate to ⁇ v ⁇ 3 integrin activation status.
- A-Q Effect of integrin knockdown on VEGFR-2 expression was evaluated by transfecting HUVECs with siRNA specific for (A) ⁇ l , (B) ⁇ 3, or (C) ⁇ 5 integrin. Cell Iy sates were analyzed for expression of VEGFR-2. Densitometry analysis was performed and results are shown as bar graphs (lower panels).
- D-E ⁇ v ⁇ 3 integrin activation dependent phosphorylation of VEGFR-2.
- HUVECs were transfected with ⁇ l, ⁇ 3, or ⁇ 5 integrin-specific siRNA and induced with 20 ng/mL VEGF for 5 min.
- D Cell lysates were analyzed for phosphorylation of VEGFR-2 using specific antibody. Densitometry analysis was performed and results are shown as bar graphs (lower panel).
- E HUVECs were incubated with ⁇ v ⁇ 3 integrin-activating antibody (Libs-1, AP-7.3, CRC-54) or ⁇ 3 integrin blocking antibody. These cells were induced with VEGF for 5 min and cell lysates were analyzed for phosphorylation of VEGFR-2 using specific antibody. Densitometry analysis was performed and results are shown as bar graphs (lower panel).
- Fig. 14 illustrates (A) photographs of endothelial cell tube formation in a matrigel assay for endothelial cells subjected to VEGF, a peptide in accordance with the present invention, and VEGF in conjunction with a peptide in accordance with the present invention; and (B) a graph showing the results.
- Fig. 15 are blots illustrating the phosphorylation status of ⁇ 3 integrin upon treatment with LM609 antibody (vitaxin).
- Fig. 16 illustrates photographs of matrigel plugs in accordance with an aspect of the invention.
- Fig. 17 illustrates a graph showing that ⁇ 3 cytoplasmic tail peptide inhibits angiogenesis in vivo.
- Fig. 18 illustrates phosphorylation ⁇ 3 integrin cytoplasmic tyrosine motifs is crucial for VEGF induced VEGFR-2 activation.
- HUVECs were either kept in suspension (lane 1 and 2) or plated on vitronectin (lane 3 and 4), laminin (lane 5 and 6), collagen (lane 7 and 8). These cells were induced with 20 ng/mL VEGF and cell lysates were analyzed for phosphorylation of cytoplasmic tyrosine motifs on ⁇ 3 integrin and p- VEGFR-2 using specific antibodies. Cell lysates were also analyzed for equal levels of ⁇ 3 integrin (middle panel) and VEGFR-2 (lower panel) as loading controls.
- Fig. 19 illustrates VEGF differentially induces SFK interactions with ⁇ 3 integrin and VEGFR-2.
- A-B Semiconfluent serum-starved HUVECs were induced with 20 ng /mL VEGF for 10 min. Cell lysates were immunoprecipitated with anti-VEGFR2 (A) or anti- ⁇ 3 integrin (B) antibody. Immunocomplexes were resolved by SDSPAGE and analyzed for Src, Yes and Fyn using specific antibodies (lane 2 and 3). HUVECs lysate was used as positive control (lane 1). Densitometry analysis was performed and fold changes were indicated (lower panel A and B).
- VEGF induced ⁇ 3 integrin phosphorylation follows c-Src phosphorylation. Serum-starved HUVECs were induced with 20ng/mL VEGF for 0-60 min. A portion of cell lysate was analyzed for activation phosphorylation of c-Src (Tyr-416) using specific antibody (upper panel C). Another portion of the cell lysate was immunoprecipitated with anti- ⁇ 3 integrin antibody and immunoblotted with antiphosphotyrosine antibody (middle lower panel C). Densitometry analysis was performed and fold changes over control are indicated (lower panel C).
- Fig. 20 illustrates c-Src directly phosphorylates ⁇ 3 integrin on cytoplasmic tyrosine motifs.
- HUVECs were either kept in suspension (lane 1) or in adhesion (lane 2 and 3) in presence of SU6656 (lane 4). HUVECs were also transfected with wild-type Src (lane 5 and 6), dominant negative Src (lane 7 and 8), or catalytically-active Src (lane 9 and 10) and these cells were induced with 20 ng/mL VEGF. Cell lysates were resolved by SDS-PAGE and analyzed for phosphorylation of ⁇ 3 integrin using specific antibodies (upper and middle panel).
- FIG. 21 illustrates c-Src-dependent ⁇ 3 integrin cytoplasmic tyrosine motifs phosphorylation required for ⁇ 3 integrin/VEGFR-2 interaction and ⁇ 3 integrin activation and ligand binding.
- A-B HUVECs treated with various pharmacological inhibitors or over expressed with various Src constructs were surface labeled with membrane-impermeable sulpho-NHS biotin and these cells were induced with 20 ng/mL VEGF.
- Cell lysates were immunoprecipitated with anti- ⁇ 3 integrin antibody and tmmunoblotted with anti-streptavidin antibody (upper panel A).
- KUVECs were induced with VEGF and cell lysates were immunoprecipitated with anti- ⁇ 3 integrin antibody and immunoblotted with anti-VEGFR-2 antibody (upper panel B). Blots were also analyzed for ⁇ 3 integrin as loading control (lower panel A and B).
- C-D Wild type and DiYF mouse lung microvascular endothelial cells were stimulated with 20 ng /mL VEGF. Cell lysates were analyzed for phosphorylation of ⁇ 3 integrin using specific antibodies (upper and middle panel of C). Wild type and DiYF mouse lung microvascular endothelial cells were transfected with DN Src and CA Src constructs and plated on vitronectin-coated plates.
- HUVECs were transiently transfected with various Src constructs. These cells were stimulated with VEGF and incubated with WOW-I Fab fragments (panel E) or FITC-fibrinogen (panel F) for 30 min at 37°C. Cells were fixed, washed, and analyzed by flow cytometry. Bars represent mean fluorescence intensity of three independent experiments performed in triplicate (panel E and F).
- Fig. 22 illustrates c-Src mediated phosphorylation of ⁇ 3 integrin cytoplasmic tyrosine motifs required for ⁇ v ⁇ 3 integrin outside-in signaling.
- A-B Src-H-, SYF, SYF+Src cells were washed and plated on vitronectin-, collagen-, or laminin-coated plates. Numbers of attached cells per field were counted. Number of Src++ cells adhered on vitronectin was assigned a value of 100%.
- C HUVECs were either transfected with various forms of Src or treated with SU6656. These cells were suspended on vitronectin-coated plates.
- Fig. 23 illustrates c-Src dependent phosphorylation of ⁇ 3 integrin cytoplasmic tyrosine motifs required for ⁇ v ⁇ 3 integrin-dependent directional migration of endothelial cells.
- A-B HUVECs (panel A) or wild type and DiYF mouse lung microvascular endothelial cells ⁇ panel B) were transfected with various forms of Src. These cells were seeded on vitronectin-coated upper wells of Boyden-type migration chamber. Cells were allowed to migrate and non-migrated cells adhered to the top surface were removed. Migrated cells were stained and number of cells per field was counted. Numerical values are represented as bar diagram and fold changes over control are indicated.
- C Wild type and DiYF mouse lung microvascular endothelial cells were grown on vitronectin-, laminin or collagen-coated plates. A wound was created across the cell monolayer by scraping away a swath of cells.
- Representative cell paths are shown tracked by video-lapse microscopy in presence of 20 ng/mL VEGF over period of 10 h. Cell paths are reconstituted such that all paths starts from origin. Unit of measures on axes is ⁇ m/h. Asterisks indicate significant difference over WT-Src transfected cells (P ⁇ 0.039).
- FIG. 24 illustrates ⁇ 3 integrin cytoplasmic tyrosine phosphorylation is required for angiogenesis in vitro and in vivo.
- A-B Wild type and DiYF mouse lung microvascular endothelial cells were transfected with various forms of Src. These cells were transferred to Matrigel-coated plates and incubated in presence of VEGF for 8 h.
- A Three random fields were photographed using phase-contrast microscope.
- B Length of tubes in random fields from each well was analyzed using ImagePro software.
- the present invention relates generally to a method of modulating angiogenesis in a tissue, and thereby affecting events in the tissue that depend on angiogenesis.
- the method comprises administering to the tissue a therapeutically effective amount of an agent that modulates (e.g., inhibits or stimulates) complex formation of Ovp 3 integrin and VEGFR2.
- VEGF stimulation via activation of it major receptor VEGFR2 leads to the tyrosine phosphorylation of Oy ⁇ 3 integrin and integrin activation.
- tyrosine residues within ⁇ v p 3 integrin are essential for complex formation with VEGFR2 and for sustained activation of VEGFR2.
- Impaired or inhibited ⁇ 3 tyrosine phosphorylation leads to reduced complex formation, reduced VEGFR2 activation, and inhibited angiogenesis.
- the inhibition of ⁇ v ⁇ 3 integrin and VEGFR2 complex formation suppresses the function of both receptors. This is exemplified by reduced angiogenesis in mice with impaired ⁇ 3 tyrosine phosphorylation.
- a similar complex formation between otvp 3 integrin and VEGFR2 also occurs on tumor cells to promote tumor angiogenesis.
- the inhibition of the complex formation of ⁇ v ⁇ 3 integrin and VEGFR2 can be used to suppress angiogenesis in endothelial cells and tumor-induced angiogenesis.
- the agents in accordance with the present invention can potentially suppress tumor cell-proliferation and metastatic activity.
- ⁇ v ⁇ 3 integrin and VEGFR2 occurs only on cells stimulated with VEGF or exposed to certain integrin ligands (e.g., vitronectin). Stimulation and over stimulation with VEGF is associated with pathological conditions, such as tumor angiogenesis. On normal quiescent endothelial cells ⁇ v ⁇ 3 integrin may be expressed but it does not complex with VEGFR2.
- the method of the present invention can target activated endothelial cells associated with pathological conditions (e.g., pathological or aberrant angiogenesis) and tumor cells without targeting normal quiescent endothelial cells. This is in contrast to agents, such as VITAXIN, that block ligand binding to ⁇ v ⁇ 3 integrin or VEGFR2.
- One aspect of the present invention therefore relates to a method of inhibiting pathological angiogenesis by administering a therapeutically effective amount of agent that substantially inhibits complex formation of ⁇ v ⁇ 3 integrin and VEGFR2 but does not block or inhibit binding of natural ligands to VEGFR2 and Ov ⁇ 3 integrin.
- agent that substantially inhibits complex formation of ⁇ v ⁇ 3 integrin and VEGFR2 but does not block or inhibit binding of natural ligands to VEGFR2 and Ov ⁇ 3 integrin.
- inhibit includes any measurable reproducible substantial reduction in: the interaction of ⁇ v ⁇ 3 integrin and VEGFR2; angiogenesis; symptoms of diseases correlated to angiogenesis; or any other activities complex formation of ⁇ v p 3 integrin and VEGFR2 may mediate.
- a substantial reduction is a "reproducible”, i.e., consistently observed, reduction in complex formation.
- a “substantial reduction” in terms of the present application is defined as a reproducible reduction (in complex formation of Ov ⁇ 3 integrin and VEGFR2) of at least about 25%, or about 50%.
- the present method of inhibiting angiogenesis in a tissue comprises contacting a tissue in which angiogenesis is occurring, or is at risk for occurring, with a composition comprising a therapeutically effective amount of an agent that is capable of inhibiting complex formation of ⁇ v ⁇ s integrin and VEGFR2 (i.e., ⁇ v ⁇ 3 integrin/VEGFR2 complex inhibiting agent).
- a composition comprising a therapeutically effective amount of an agent that is capable of inhibiting complex formation of ⁇ v ⁇ s integrin and VEGFR2 (i.e., ⁇ v ⁇ 3 integrin/VEGFR2 complex inhibiting agent).
- the method comprises administering to a patient a therapeutically effective amount of a physiologically tolerable composition containing an agent that is an inhibitor of complex formation of ⁇ v ⁇ 3 integrin and VEGFR2.
- the dosage ranges for the administration of the agent depend upon the foim of the inhibitor, and its potency, and are amounts large enough to produce the desired effect in which angiogenesis and the disease symptoms mediated by angiogenesis are ameliorated.
- the dosage should not be so large as to cause adverse side effects.
- the dosage can also be adjusted by the individual physician in the event of any complication.
- a therapeutically effective amount is an amount of agent that is an inhibitor of complex formation of ⁇ v p3 integrin and VEGFR2 sufficient to produce a measurable inhibition of angiogenesis in the tissue being treated, i.e., an angiogenesis-inhibiting amount. Inhibition of angiogenesis can be measured in situ by immunohistochemistiy or by other methods known to one skilled in the art.
- the agent can comprise an inhibiting compound or inhibiting peptide that competes with O v p 3 integrin or VEGFR2 for interaction between the two receptors.
- the inhibiting peptide can have an amino acid sequence of about 5 to about 50 amino acids (e.g., about 10 to about 30 amino acids) that corresponds to an about 5 to about 50 amino acid portion of the amino acid sequence of the VEGFR2 or 0 ⁇ 3 integrin.
- the inhibiting peptide has an amino acid sequence with a sequence identity that is substantially homologous to a portion of the amino acid sequence of ⁇ v p 3 integrin or VEGFR2.
- substantially homologous it is meant the inhibiting peptide has at least about 70%, about 80%, about 90% or about 100% sequence identity with a portion of the amino acid sequence of 0 ⁇ 3 integrin or VEGFR2.
- the inhibiting peptide can correspond to an about 5 to about 50 amino acid portion of the extracellular domain or cytoplasmic domain of ⁇ v p3 integrin or VEGFR2.
- Particular peptides include those that correspond to a portion of 0 ⁇ 3 integrin or VEGFR2 domain that comprises a tyrosine residue. It was found that phosphorylation of tyrosine is required for complex formation of ⁇ ⁇ p3 integrin and VEGFR2.
- a peptide comprising an amino acid sequence that corresponds to a portion of the amino acid sequence of ⁇ v ⁇ 3 integrin or VEGFR2 containing a tyrosine residue can compete with the tyrosine residue for phosphorylation.
- the inhibiting peptide can comprise about 5 to about 30 amino acids and have substantially the same sequence identity as an about 5 to about 30 amino acid portion of the cytoplasmic domain of ⁇ 3 that includes tyrosine.
- the inhibiting peptide can comprise can have an amino acid sequence of about 5 to about 30 amino acids and correspond to (or have a sequence identity of) a portion of the cytoplasmic domain of ⁇ 3 integrin that includes tyrosine 747.
- Examples of peptides that can be used as an agent in accordance with the present invention can have an amino selected from the group consisting of:
- DTANNPLYKEATSTFT-COOH SEQ ID NO: 1
- YGRKKRRQRRRGDTANNPLYKEATSTFT-COOH SEQ ID NO: 2
- These peptides correspond to a portion of the cytoplasmic domain of ⁇ 3 integrin comprising the amino acid sequence DTANNPL YKEATSTFTNITYRGT. (SEQ ID NO: 3)
- peptides that can be used as an agent in accordance with the present invention and that correspond to a portion of the amino acid sequence of O v ⁇ 3 integrin can comprises an amino acid sequence selected from group consisting of: KEFAKFEEER (SEQ ID NO: 4) and ARAKWDTANN (SEQ ID NO: 5).
- Still other examples of peptides that can be used as an agent in accordance with the present invention and that correspond to a portion of the amino acid sequence of VEGRFR2 can comprises an amino acid sequence selected from group consisting of:
- CMEEEEVCDPKFHYDNTAGI SEQ ID NO: 6
- QTSGYQSGYHSDDTDITVYS SEQ ID NO: 7
- RDIYKDPDYVRKGDARLPLK SEQ ID NO: 8
- WMAPETIFDRVYTIQSDVWSFGV SEQ ID NO: 9
- LGASPYPGVKIDEEFCRRLK SEQ ID NO: 10
- EGTRMRAPDYTTPEMYQTML SEQ ID NO: 11
- GNLLQANAQQDGKDYrVLPISETLSMEEDS SEQ ID NO: 12
- peptides in accordance with the present invention can be subject to various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use.
- a peptide that is an inhibitor of complex formation of Ov ⁇ 3 integrin and VEGFR2 corresponds to, rather than is identical to, the sequence of a recited peptide where one or more changes are made and it retains the ability to function as an inhibitor of complex formation of ⁇ v p 3 integrin and VEGFR2.
- a peptide can be in any of a variety of forms of peptide derivatives, that include amides, conjugates with proteins, cyclized peptides, polymerized peptides, analogs, fragments, chemically modified peptides, and the like derivatives.
- analog includes any peptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and that is an inhibitor of complex formation of ⁇ v p 3 integrin and VEGFR2 as described herein.
- conservative substitutions include the substitution of one non-polar (hydrophobic) residue, such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
- the phrase "conservative substitution” also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite inhibition activity.
- “Chemical derivative” refers to a subject peptide having one or more residues chemically derivatized by reaction of a functional side group.
- Such derivatized molecules include for example, those molecules in which free-amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
- Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
- Free hydroxyl groups may be derivatized to form 0-acyl or O-alkyl derivatives.
- the imidazole nitrogen of histidine may be derivatized to form N-tm-benzylhistidine.
- chemical derivatives those peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.
- 4-hydroxyproline may be substituted for proline
- 5-hydroxylysine may be substituted for lysine
- 3-methylhistidine may be substituted for histidine
- homoserine may be substituted for serine
- ornithine may be substituted for lysine.
- Peptides of the present invention also include any peptide having one or more additions and/or deletions or residues relative to the sequence of a peptide whose sequence is shown herein, so long as the requisite activity is maintained.
- fragment refers to any subject peptide having an amino acid residue sequence shorter than that of a peptide whose amino acid residue sequence is shown herein.
- Additional residues may also be added at either terminus of a peptide for the purpose of providing a "linker" by which the peptides of this invention can be conveniently affixed to a label or solid matrix, or carrier.
- Labels, solid matrices and carriers that can be used with the polypeptides of this invention are described herein below.
- Amino acid residue linkers are usually at least one residue and can be 40 or more residues, more often 1 to 10 residues. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.
- a subject polypeptide can differ by the sequence being modified by terminal-NH 2 acylation, e.g., acetylation, or thioglycolic acid amidation, by terminal-carboxylamidation, e.g., with ammonia, methylamine, and the like terminal modifications. Terminal modifications are useful, as is well known, to reduce susceptibility by proteinase digestion, and therefore serve to prolong half life of the polypeptides in solutions, particularly biological fluids where proteases may be present.
- polypeptide cyclization is also a useful terminal modification, and is particularly preferred also because of the stable structures formed by cyclization and in view of the biological activities observed for such cyclic peptides as described herein.
- Acids which are capable of forming salts with the peptides of the present invention, include inorganic acids such as trifluoroacetic acid (TFA) hydrochloric acid (HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid or the like.
- TFA trifluoroacetic acid
- HCl hydrochloric acid
- hydrobromic acid perchloric acid
- nitric acid nitric acid
- thiocyanic acid sulfuric acid
- sulfuric acid phosphoric acetic acid
- propionic acid glycolic acid
- lactic acid pyruvic acid
- Bases capable of forming salts with the peptides of the present invention include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (e.g. ethanolamine, diethanolamine and the like).
- inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like
- organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (e.g. ethanolamine, diethanolamine and the like).
- a peptide of the present invention also referred to herein as a subject peptide, can be synthesized by any of the techniques that are known to those skilled in the polypeptide art, including recombinant DNA techniques. Synthetic chemistry techniques, such as a solid-phase Merrifield-type synthesis, can be used for reasons of purity, antigenic specificity, freedom from undesired side products, ease of production and the like. A summary of the many techniques available can be found in Steward et al. 5 "Solid Phase Peptide Synthesis", W. H. Freeman Co., San Francisco, 1969; Bodanszky, et al., "Peptide Synthesis", John Wiley & Sons, Second Edition, 1976; J.
- the solid-phase synthesis methods contemplated comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine.
- the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group.
- the protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support.
- the protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to afford the final linear polypeptide.
- the agent that inhibits complex formation of ⁇ v ⁇ 3 integrin and VEGFR2 can comprise a c-SRC kinase inhibitor. It was found that ⁇ v ⁇ 3 integrin tyrosine phosphorylation is crucial for VEGF tyrosine phosphorylation of VEGFR2 and the ⁇ v ⁇ 3 integrin tyrosine phosphorylation is directly mediated by c-SRC kinase, which is able to phosphorylate the cytoplasmic domain of ⁇ v p3.
- the c-SRC inhibitor can comprise a 3-(4,5,6,7- tetrahydroinol-2-ylmethylidene)-2-indolinone derivative that inhibits 0 ⁇ 3 cytoplasmic phosphorylation.
- 3-(4,5,6,7-tetrahydroinol-2- ylmethylidene) -2-indolinone derivatives include compounds having the following general formula:
- Ri is -S(O) n R2 (where n is 0, 1, or 2 and R 2 is alkyl or aralkyl) or -SO 2
- R 3 and R4 are independently hydrogen, alkyl, cycloalkyl, alkoxyalkyl, or hydroxyalkyl.
- Examples of particular 3-(4,5,6,7-tetrahydroinol-2-ylmethyl ⁇ dene) -2- indolinone derivatives include 2-oxo-3(4,5,6, 7-tetrahydro-l-H-indol-2- ylmethylene)-2,3-dihydro-lH-mdole-5-sulfonic acid dimethylamide and 2-oxo-
- 3 -(4,5,6,7-tetrahydroinol-2-ylmethylidene) -2- indolinone derivatives that can be potentially used as c-SRC inhibitors to ⁇ v ⁇ 3 cytoplasmic phosphorylation include 3-(4,5,6,7-tetrahydromol-2-ylmethylidene) - 2-indolinone derivatives disclosed in U.S. Patent No. 6,777,417, which is herein incorporated by reference in its entirety.
- c-SRC inhibitors that can potentially be used in accordance with the present invention include c- SRC inhibitors disclosed in, for example, PCT application WO 01/00214 and US Patent Publication 2006/0223815, both of which are herein incorporated by reference in their entirety.
- the c-SRC inhibitor can include a biological agent that inhibits expression and/or activity of c-SRC-kinase.
- the biological agent can include an antisense oligonucleotide that inhibits expression and/or activity of the c-SRC-kinase.
- Antisense nucleotides are relatively short nucleic acids that are complementary (or antisense) to the coding strand (sense strand) of the mRNA encoding the c-SRC kinase. Although antisense oligonucleotides are typically RNA based, they can also be DNA based. Additionally, antisense oligonucleotides are often modified to increase their stability.
- oligonucleotides binding of these relatively short oligonucleotides to the mRNA of c-SRC kinase is believed to induce stretches of double stranded RNA that trigger degradation of the messages by endogenous RNAses. Additionally, sometimes the oligonucleotides are specifically designed to bind near the promoter of the message, and under these circumstances, the antisense oligonucleotides may additionally interfere with translation of the message. Regardless of the specific mechanism by which antisense oligonucleotides function, their administration to a cell or tissue allows the degradation of the mRNA encoding the c-SRC kinase.
- antisense oligonucleotides decrease the expression and/or activity of the c-SRC kinase.
- the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
- the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
- the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.
- oligonucleotide may be conjugated to another molecule.
- the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytos ⁇ ie, 5-(carboxyhydroxytriethyl)uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminoinethylura- cil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3- methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl
- the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2- fluoroarabinose, xylulose, and hexose.
- the antisense oligonucleotide can also contain a neutral peptide4ike backbone.
- peptide nucleic acid (PNA)-oligomers are termed peptide nucleic acid (PNA)-oligomers and are described, e.g., m Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:14670 and in Eglom et al. (1993) Nature 365:566.
- PNA peptide nucleic acid
- One advantage of PNA oligomers is their capability to bind to complementary DNA essentially independently from the ionic strength of the medium due to the neutral backbone of the DNA.
- the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoram ⁇ dothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester , and a formacetal or analog thereof.
- the antisense oligonucleotide is an - anomeric oligonucleotide.
- oligonucleotide forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual -units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).
- the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al.,
- Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
- an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
- phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209)
- methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al.,
- oligonucleotide can be readily performed by one of skill in the art. Given the nucleic acid sequence encoding a c- SRC kinase, one of skill in the art can design antisense oligonucleotides that bind to the mRNA encoding the SRC-kinase, and test these oligonucleotides in an in vitro or in vivo system to confirm that they bind to and mediate the degradation of the mRNA encoding the particular protein.
- an antisense oligonucleotide that specifically binds to and mediates the degradation of the SRC-kinase, it is important that the sequence recognized by the oligonucleotide is unique or substantially unique to the SRC-kinase. For example, sequences that are frequently repeated across the SRC-kinase may not be an ideal choice for the design of an oligonucleotide that specifically recognizes and degrades a particular message.
- One of skill in the art can design an oligonucleotide, and compare the sequence of that oligonucleotide to nucleic acid sequences that are deposited in publicly available databases to confirm that the sequence is specific or substantially specific for the SRC-kinase.
- antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.
- modified antisense molecules designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.
- a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA.
- a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
- Such vectors can be constructed by recombinant DNA technology methods standard in the art.
- Vectors can be plasm ⁇ d, viral, or others known in the art, used for replication and expression in mammalian cells.
- Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive.
- Such promoters include but are not limited to: the SV40 early promoter region (Bemoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U. S. A. 78:1441- 1445), the regulatory sequences of the metallothionein gene (Brinster et al, 1982, Nature 296:39-42), etc.
- the SV40 early promoter region Bemoist and Chambon, 1981, Nature 290:304-310
- the promoter contained in the 3' long terminal repeat of Rous sarcoma virus Yamamoto et al., 1980, Cell 22:787-797
- plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site.
- viral vectors can be used which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systematically).
- the c-SRC inhibitor can also include RNAi constructs that can specifically block expression of a c-SRC gene.
- RNA interference or "RNAi” is a term initially applied to a phenomenon observed in plants and worms where double-stranded RNA (dsRNA) blocks gene expression in a specific and post- transcriptional manner. Without being bound by theory, RNAi appears to involve mRN A degradation, however the biochemical mechanisms are currently an active area of research. Despite some mystery regarding the mechanism of action, RNAi provides a useful method of inhibiting gene expression in vitro or in vivo.
- dsRNA refers to siRNA molecules, or other RNA molecules including a double stranded feature and able to be processed to siRNA in cells, such as hairpin RNA moieties.
- RNAi refers to (indicates) the ability to distinguish which RNAs are to be degraded by the RNAi process, e.g., degradation occurs in a sequence-specific manner rather man by a sequence- independent dsRNA response.
- RNAi construct is a generic term used throughout the specification to include small interfering RNAs (siRNAs), hairpin RNAs, and other RNA species which can be cleaved in vivo to form siRNAs.
- RNAi constructs herein also include expression vectors (also referred to as RNAi expression vectors) capable of giving rise to transcripts which form dsRNAs or hairpin RNAs in cells, and/or transcripts which can produce siRNAs in vivo.
- RNAi expression vector refers to replicable nucleic acid constructs used to express (transcribe) RNA which produces siRNA moieties in the cell in which the construct is expressed.
- Such vectors include a transcriptional unit comprising an assembly of (I) genetic element(s) having a regulatory role in gene expression, for example, promoters, operators, or enhancers, operatively linked to (2) a "coding" sequence which is transcribed to produce a double-stranded RNA (two RNA moieties that anneal in the cell to form an siRNA, or a single hairpin RNA which can be processed to an siRNA), and (3) appropriate transcription initiation and termination sequences.
- promoter and other regulatory elements generally varies according to the intended host cell.
- expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to ctrcular double stranded DNA loops which, in their vector form are not bound to the chromosome.
- plasmid and vector are used interchangeably as the plasmid is the most commonly used form of vector.
- the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
- RNAi constructs contain a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript for the c-SRC gene to be inhibited (i.e., the "target" gene).
- the double-stranded RNA need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi.
- the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence.
- the number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center of the siRNA duplex are most critical and may essentially abolish cleavage of the target RNA. In contrast, nucleotides at the 3' end of the siRNA strand that is complementary to the target RNA do not significantly contribute to specificity of the target recognition.
- Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991 , and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred.
- the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript.
- RNAi constructs can be carried out by chemical synthetic methods or by recombinant nucleic acid techniques. Endogenous RNA polymerase of the treated cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vitro.
- the RNAi constructs may include modifications to either the phosphate-sugar backbone or the nucleoside, e.g., to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and/or change other pharmacokinetic properties.
- the phosphodiester linkages of natural RNA may be modified to include at least one of an nitrogen or sulfur heteroatom.
- RNA structure may be tailored to allow specific genetic inhibition while avoiding a general response to dsRNA.
- bases may be modified to block the activity of adenosine deaminase.
- the RNAi construct may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.
- RNAi constructs see, for example, Heidenreich e t a 1. (1997) Nucleic Acids Res, 25:776-780; Wilson et al. (1994) J MoI Recog 7:89-98; Chen et al. (1995) Nucleic Acids Res 23:2661-2668; Hirschbein et al. (1997) Antisense Nucleic Acid Drug Dev 7:55-61).
- RNAi construct can be modified with phosphorothioates, phosphoramidate, phosphodithioates, chimeric methylphosphonate-phosphodie- sters, peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g., T- substituted ribonucleosides, a-configuration).
- the double-stranded structure may be formed by a single self- complementary RNA strand or two complementary RNA strands.
- RNA duplex formation may be initiated either inside or outside the cell.
- the RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition, while lower doses may also be useful for specific applications. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition
- the subject RNAi constructs are "small interfering RNAs" or "siRNAs.” These nucleic acids are around 19-30 nucleotides in length, and even more preferably 21-23 nucleotides in length, e.g., corresponding in length to the fragments generated by nuclease "dicing" of longer double-stranded RNAs.
- the siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex.
- the 21-23 nucleotides siRNA molecules comprise a 3' hydroxyl group.
- siRNA molecules of the present invention can be obtained using a number of techniques known to those of skill in the art.
- the siRNA can be chemically synthesized or recombinantly produced using methods known in the art.
- short sense and antisense RNA oligomers can be synthesized and annealed to form double-stranded RNA structures with 2-nucleotide overhangs at each end (Caplen, et al. (2001) Proc Natl Acad Sci USA, 98:9742-9747; Elbashir, et al. (2001) EMBO J, 20:6877-88).
- These double-stranded siRNA structures can then be directly introduced to cells, either by passive uptake or a delivery system of choice, such as described below.
- the siRNA molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify siRNAs. Alternatively, non-denaturing methods, such as non-denaturing column chromatography, can be used to purify the siRNA. In addition, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, affinity purification with antibody can be used to purify siRNAs. [00112] In certain preferred embodiments, at least one strand of the siRNA molecules has a 3 1 overhang from about 1 to about 6 nucleotides in length, though may be from 2 to 4 nucleotides in length.
- the 3' overhangs are 1-3 nucleotides in length, In certain embodiments, one strand having a 3' overhang and the other strand being blunt-ended or also having an overhang.
- the length of the overhangs may be the same or different for each strand.
- the 3' overhangs can be stabilized against degradation.
- the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
- substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine nucleotide 3' overhangs by 2'-deoxythyinidine is tolerated and does not affect the efficiency of RNAi.
- the absence of a 2' hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium and may be beneficial in vivo.
- the RNAi construct is in the form of a long double-stranded RNA.
- the RNAi construct is at least 25, 50, 10O 5 200, 300 or 400 bases.
- the RNAi construct is 400-800 bases in length.
- the double-stranded RNAs are digested intracellularly, e.g., to produce siRNA sequences in the cell.
- use of long double- stranded RNAs in vivo is not always practical, presumably because of deleterious effects, which may be caused by the sequence-independent dsRN A response.
- the use of local delivery systems and/or agents which reduce the effects of interferon or PKR are preferred.
- the RNAi construct is in the form of a hairpin structure (named as hairpin RNA).
- hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Examples of making and using such hairpin RNAs for gene silencing in mammalian cells are described in, for example, Paddison et al., Genes Dev, 2002, 16:948-58; McCaffrey et al. Nature, 2002, 418:38-9; McManus et al., RNA 5 2002, 8:842-50; Yu et al, Proc Natl Acad Sci USA, 2002, 99:6047-52).
- hairpin RNAs are engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell.
- a plasmid is used to deliver the double- stranded RNA, e.g., as a transcriptional product.
- the plasmid is designed to include a "coding sequence" for each of the sense and antisense strands of the RNAI construct.
- the coding sequences can be the same sequence, e.g., flanked by inverted promoters, or can be two separate sequences each under transcriptional control of separate promoters. After the coding sequence is transcribed, the complementary RNA transcripts base-pair to form the double-stranded RNA.
- PCT application WOO 1 /77350 describes an exemplary vector for bidirectional transcription of a transgene to yield both sense and antisense RNA transcripts of the same transgene in a eukaryotic cell.
- the present invention provides a recombinant vector having the following unique characteristics: it comprises a viral replicon having two overlapping transcription units arranged in an opposing orientation and flanking a transgene for an RNAi construct of interest, wherein the two overlapping transcription units yield both sense and antisense RNA transcripts from the same transgene fragment in a host cell.
- RNAi constructs can comprise either long stretches of double stranded RNA identical or substantially identical to the target nucleic acid sequence or short stretches of double stranded RNA identical to substantially identical to only a region of the target nucleic acid sequence. Exemplary methods of making and delivering either long or short RNAi constructs can be found, for example, in WO01/68836 and WO01/75164.
- RNAi constructs that specifically recognize a particular gene, or a particular family of genes can be selected using methodology outlined in detail above with respect to the selection of antisense oligonucleotide.
- methods of delivery RNAi constructs include the methods for delivery antisense oligonucleotides outlined in detail above.
- the c-SRC inhibitors of the present invention can also include ribozymes molecules designed to catalytically cleave mRNA transcripts to prevent translation of mRNA (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225 and U.S. Pat. No. 5,093,246). While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy particular mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA.
- target mRNA have the following sequence of two bases: 5'-UG-3'.
- the construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.
- the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one which occurs naturally in Tetrahymena thermophila ⁇ known as the IVS, or L- 19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-433; published International patent application No. WO88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216).
- Cech-type ribozymes such as the one which occurs naturally in Tetrahymena thermophila ⁇ known as the IVS, or L- 19 IVS RNA
- the Cech-type ribozymes have an eight base pair active site that hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
- the invention encompasses those Cech-type ribozymes that target eight base-pair active site sequences.
- the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and can be delivered to cells in vitro or in vivo.
- a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy targeted messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
- the c-SRC kinase inhibitors can also include antibodies used as inhibitors of the activity of c-SRC kinase activity.
- Antibodies can have extraordinary affinity and specificity for particular epitopes. Antibodies that bind to a particular protein in such a way that the binding of the antibody to the epitope on the protein can interfere with the iunction of that protein. For example, an antibody may inhibit the function of the protein by sterically hindering the proper protein-protein interactions or occupying active sites. Alternatively, the binding of the antibody to an epitope on the particular protein may alter the conformation of that protein such that it is no longer able to properly function.
- the antibody may bind to a different site on the enzyme to sterically hinder the protein- protein interactions required for enzyme function.
- the antibody may bind to a different site on the enzyme and alter the conformation of the enzyme such that the enzyme is no longer able to function.
- Monoclonal or polyclonal antibodies can be made using standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
- a mammal such as a mouse, a hamster, a rat, a goat, or a rabbit can be immunized with an immunogenic form of the peptide.
- lymphocytes antibody-producing cells
- Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a particular polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
- antibodies can be screened and tested to identify those antibodies that can inhibit the function of the SRC-kinase.
- One of skill in the art will recognize that not every antibody that is specifically immunoreactive with the SRC-kinase will interfere with the function of that protein. However, one of skill in the art can readily test antibodies to identify those that are capable of blocking the function of a particular protein.
- the term antibody as used herein is intended to include fragments thereof which are also specifically reactive with a particular polypeptide.
- Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab) 2 fragments can be generated by treating antibody with pepsin.
- the resulting F(ab) 2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.
- the antibody of the present invention is further intended to include bispecific and chimeric molecules having affinity for a particular protein conferred by at least one CDR region of the antibody.
- Both monoclonal and polyclonal antibodies (Ab) directed against a particular polypeptides, and antibody fragments such as Fab, F(ab) 2 , Fv and scFv can be used to block the action of a particular protein.
- Such antibodies can be used either in an experimental context to further understand the role of a particular protein in a biological process, or in a therapeutic context.
- the present invention also relates to an agent that stimulates complex formation of Ov ⁇ 3 integrin and VEGFR2 on endothelial cells expressing Oyp 3 uitegrin and VEGFR2.
- ang ⁇ ogenesis of tissues can be increased, stimulated, or promoted by the adminsitration of an agent that stimualtes complexing of Ov ⁇ 3 integrin and VEGFR2 (i.e ; ⁇ v ⁇ 3 integrin/VEGFR2 complex stimulating agent).
- the term “stimulate”, “stimulating” or “stimulant” includes any measurable reproducible increase in: speed, duration, or degree in the interaction of ⁇ v p3 integrin and VEGFR2; ang ⁇ ogenesis; or any other activities complex formation of ⁇ v ⁇ 3 integrin and VEGFR2 may mediate.
- a population of cells or tissue that express O v ⁇ 3 integrin and VEGFR2, such as endothelial cells, can be contacted with a therapeutically effective amount of a ⁇ v ⁇ 3 integrin/VEGFR2 complex stimulating agent under conditions effective to stimulate complex formation of ⁇ v ⁇ 3 integrin and VEGFR2.
- the stimulation of the complex formation of ⁇ v ⁇ 3 integrin and VEGFR2 can be used to increase angiogenesis in endothelial cells and promote angiogenesis in, for example, ischemic tissue.
- the ⁇ v ⁇ 3 integrin/VEGFR2 complex stimulating agent can stimulate complex formation of ⁇ v ⁇ 3 integrin and VEGFR2 on cells stimulated not stimulated with VEGF or exposed to certain integrin ligands (e.g., vitronectin).
- the stimulation of angiogenesis can play an important role in a variety of physiological processes such as embryonic development, wound healing, organ regeneration and female reproductive processes such as follicle development in the corpus Iuteum during ovulation and placental growth after pregnancy. Additionally, millions of patients per year in the U.S. suffer from myocardial infarction (MI) and/or critical limb ischemia. Many millions more suffer from related syndromes due to atherosclerosis. Many of these patients will benefit from the ability to stimulate angiogenesis in ischemic areas.
- MI myocardial infarction
- critical limb ischemia Many millions more suffer from related syndromes due to atherosclerosis. Many of these patients will benefit from the ability to stimulate angiogenesis in ischemic areas.
- compositions are to be used for therapeutic purposes, the dose(s) and route of administration will depend upon the nature of the patient and condition to be treated, and will be at the discretion of the attending physician or veterinarian. Suitable routes include oral, subcutaneous, intramuscular, intraperitoneal or intravenous injection, parenteral, topical application, implants etc.
- the agent can comprise a stimulating compound or stimulating peptide that promotes complexing of O v ⁇ 3 integrin and VEGFR2.
- the stimulating peptide can have an amino acid sequence of about 5 to about 50 amino acids (e.g., about 10 to about 30 amino acids) that corresponds to an about 5 to about 50 amino acid portion of the amino acid sequence of the VEGFR2 or ⁇ v p3 integrin.
- the inhibiting peptide has an amino acid sequence with a sequence identity that is substantially homologous to a portion of the amino acid sequence of ctv ⁇ 3 integrin or VEGFR2.
- substantially homologous it is meant the inhibiting peptide has at least about 70%, about 80%, about 90% or about 100% sequence identity with a portion of the amino acid sequence of ⁇ v ⁇ 3 integrin or VEGFR2.
- the stimulating peptide can have an amino acid sequence identity of YGRKKRRQRRRGKEATSTFTMTYRGT-COOH (SEQ ID NO: 13) or KEATSTFTMTYRGT-COOH (SEQ ID NO: 15). It was found that these peptides promote angiogenesis of epithelial cells expressing ⁇ v ⁇ 3 integrin and VEGFR2 both in the presence and the absence of VEGF.
- the peptide in accordance with the present invention can be subject to various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use.
- the peptide can be in any of a variety of forms of peptide derivatives, that include amides, conjugates with proteins, cyclized peptides, polymerized peptides, analogs, fragments, chemically modified peptides, and the like derivatives.
- the O v ⁇ 3 integrin/VEGFR2 stimulating can comprise c-SRC kinase and/or an agent that up-regulates expression of the c-SRC kinase. It was found that ⁇ v ⁇ 3 integrin tyrosine phosphorylation is directly mediated by c-SRC kinase, which is able to phosphorylate the cytoplasmic domain of ⁇ v p3.
- c-SRC via direct phosphorylation of ⁇ v ⁇ 3 integrin cytoplasmic motif controls functional association between Ct ⁇ 3 and VEGFR2, which in turn regulates activation of both receptors on endothelial cells.
- the administration of c-SRC kinase an or agent that upregulates expression of c-SRC kinase to endothelial cells expressing 0 ⁇ 3 and VEGFR2 can promote angiogenesis.
- the ⁇ x v p 3 integrin/VEGFR2 complex inhibiting/stimulating (i.e., modulating) agents in accordance with the present invention can be provided in a pharmaceutical compositions.
- compositions will generally comprise an effective amount of agent, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Combined therapeutics are also contemplated, and the same type of underlying pharmaceutical compositions may be employed for both single and combined medicaments.
- phrases "pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
- Veterinary uses are equally included within the invention and "pharmaceutically acceptable” formulations include formulations for both clinical and/or veterinary use.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
- the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
- preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards. Supplementary active ingredients can also be incorporated into the compositions.
- "Unit dosage" formulations are those containing a dose or sub-dose of the administered ingredient adapted for a particular timed delivery.
- exemplary "unit dosage'Tormulations are those containing a daily dose or unit or daily sub-dose or a weekly dose or unit or weekly sub-dose and the like.
- the O v p 3 integrin/VEGFR2 complex modulating agents of the present invention can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, transdermal, intravitreal, or other such routes, including peristaltic administration and direct instillation into a tumor or disease site (intracavity administration).
- compositions that contains such an ⁇ v ⁇ 3 integrin/VEGFR2 complex modulating agent as an active ingredient will be known to those of skill in the art in light of the present disclosure.
- such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
- compositions of the ⁇ v ⁇ 3 integrin/VEGFR2 complex modulating agents can be formulated into a sterile aqueous composition in a neutral or salt form.
- Solutions as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
- Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein), and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, trifluoroacetic, oxalic, tartaric, mandelic, and the like.
- Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
- Examples of carriers include solvents and dispersion media containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- solvents and dispersion media containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- isotonic agents for example, sugars or sodium chloride.
- the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants.
- all such preparations should contain a preservative to prevent the growth of microorganisms.
- the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
- the ⁇ v p 3 integrin/VEGFR2 complex modulating agents can be extensively dialyzed to remove undesired small molecular weight molecules, and/or lyophilized for more ready formulation into a desired vehicle, where appropriate.
- Sterile injectable solutions are prepared by incorporating the active agents in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as desired, followed by filtered sterilization.
- dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above.
- compositions in accordance with the invention will generally include an amount of the O v p 3 integrin/VEGFR2 complex modulating agent admixed with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give a range of final concentrations, depending on the intended use.
- an acceptable pharmaceutical diluent or excipient such as a sterile aqueous solution
- polypeptide or conjugate solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
- Formulations of the O v P 3 ⁇ ntegrin/VEGFR2 complex modulating agents are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but other pharmaceutically acceptable forms are also contemplated, e.g., tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, topical formulations, liposomal forms and the like. The type of form for administration will be matched to the disease or disorder to be treated. [00149] Pharmaceutical "slow release" capsules or “sustained release” compositions or preparations may be used and are generally applicable.
- Slow release formulations are generally designed to give a constant drug level over an extended period and may be used to deliver ci v ⁇ 3 integrin/VEGFR2 complex modulating agents in accordance with the present invention.
- the slow release formulations are typically implanted in the vicinity of the disease site, for example, at the site of a tumor.
- sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide or immunoconjugate, which matrices are in the form of shaped articles, e.g., films or microcapsule.
- sustained-release matrices include polyesters; hydrogels, for example, poly(2-hydroxyethyl-methacrylate) or ⁇ oly(vinylalcohol); polylactides, e.g., U.S. Pat. No. 3,773,919; copolymers of L-glutamic acid and .gamma.
- ethyl-L-glutamate ethyl-L-glutamate
- non-degradable ethylene- vinyl acetate degradable lactic acid-glycol ⁇ c acid copolymers, such as the Lupron Depot (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate); and poly-D-(-)-3-hydroxybutyric acid.
- Lupron Depot injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
- poly-D-(-)-3-hydroxybutyric acid poly-D-(-)-3-hydroxybutyric acid.
- polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days
- certain hydrogels release proteins for shorter time periods.
- encapsulated polypeptides remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, thus reducing biological activity and/or changing immunogenicity. Rational strategies are available for stabilization depending on the mechanism involved.
- the aggregation mechanism involves intermolecular S-S bond formation through thio-disulfide interchange
- stabilization is achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, developing specific polymer matrix compositions, and the like.
- liposomes and/or nanoparticles may also be employed with the 0, ⁇ 3 integrin/VEGFR2 complex modulating agents.
- the formation and use of liposomes is generally known to those of skill in the art, as summarized below.
- Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
- MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 , containing an aqueous solution in the core.
- SUVs small unilamellar vesicles
- Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios, the liposome is the preferred structure.
- the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
- Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell- surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one may operate at the same time.
- Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 ⁇ m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl- cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made. [00157] Many diseases with an angiogenic component are associated with the eye.
- diseases associated with corneal neovascularization include, but are not limited to, age- related macular degeneration, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginal keratolysis, trauma, rheumatoid arthritis,
- Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery occlusion, carotid obstructive disease, chrome uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eales disease, Bechets disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Bests disease, myopia, optic pits, Stargarts disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications.
- Ovp 3 integrin/VEGFR2 complex inhibiting agents may be advantageously employed in the preparation of pharmaceutical compositions suitable for use as ophthalmic solutions, including those for intravitreal and/or intracameral administration.
- compositions comprising ⁇ v ⁇ 3 integrin/VEGFR2 complex inhibiting agents of the invention can be administered to the eye or eyes of the subject in need of treatment in the form of an ophthalmic preparation prepared in accordance with conventional pharmaceutical practice, see for example "Remington's Pharmaceutical Sciences” 15th Edition, pages 1488 to 1501 (Mack Publishing Co., Easton, Pa.).
- the ophthalmic preparation can contain the ⁇ Xv ⁇ 3 integrin/VEGFR2 complex inhibiting agents in a pharmaceutically acceptable solution, suspension or ointment. Some variation in concentration will necessarily occur, depending on the particular compound employed, the condition of the subject to be treated and the like, and the person responsible for treatment will determine the most suitable concentration for the individual subject.
- the ophthalmic preparation will preferably be in the form of a sterile aqueous solution containing, if desired, additional ingredients, for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity-increasing agents and the like.
- Examples of preservatives for use in such a solution include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal and the like.
- Suitable buffers include boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate and the like, in amounts sufficient to maintain the pH at between about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.
- Suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, and the like, such that the sodium chloride equivalent of the ophthalmic solution is in the range 0.9 plus or minus 0.2%.
- antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfite, thiourea and the like.
- wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol.
- Suitable viscosity-increasing agents include dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose and the like.
- the ophthalmic preparation will be administered topically to the eye of the subject in need of treatment by conventional methods, for example in the form of drops or by bathing the eye in the ophthalmic solution.
- the 0 ⁇ 3 integrin/VEGFR2 complex modulating agents can be formulated for topical administration.
- Topical formulations include those for delivery via the mouth (buccal) and through the skin.
- Topical delivery systems also include transdermal patches containing the ingredient to be administered. Delivery through the skin can further be achieved by iontophoresis or electrotransport, if desired.
- Formulations suitable for topical administration in the mouth include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier.
- Formulations suitable for topical administration to the skin include ointments, creams, gels and pastes comprising the ingredient to be administered in a pharmaceutical acceptable carrier.
- O v ⁇ s integrin/VEGFR2 complex modulating agents for topical use includes the preparation of oleaginous or water-soluble ointment bases, as is well known to those in the art.
- these compositions may include vegetable oils, animal fats, and more preferably, semisolid hydrocarbons obtained from petroleum.
- Particular components used may include white ointment, yellow ointment, cetyl esters wax, oleic acid, olive oil, paraffin, petrolatum, white petrolatum, spermaceti, starch glycerite, white wax, yellow wax, lanolin, anhydrous lanolin and glyceryl monostearate.
- Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
- Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
- nasal and respiratory routes are contemplated for treating various conditions. These delivery routes are also suitable for delivering agents into the systemic circulation.
- Formulations of active ingredients in carriers suitable for nasal administration are therefore also included within the invention, for example, nasal solutions, sprays, aerosols and inhalants.
- the carrier is a solid
- the formulations include a coarse powder having a particle size, for example, in the range of 20 to 500 microns, which is administered, e.g., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
- Examples of formulations wherein the carrier is a liquid are useful in nasal administration.
- Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays and are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained.
- antimicrobial preservatives similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in the formulation.
- Various commercial nasal preparations are known and include, for example, antibiotics and antihistamines and are used for asthma prophylaxis.
- Inhalations and inhalants are pharmaceutical preparations designed for delivering a drug or compound into the respiratory tree of a patient. A vapor or mist is administered and reaches the affected area. This route can also be employed to deliver agents into the systemic circulation. Inhalations may be administered by the nasal or oral respiratory routes. The administration of inhalation solutions is only effective if the droplets are sufficiently fine and uniform in size so that the mist reaches the bronchioles.
- Another group of products also known as inhalations, and sometimes called insufflations, comprises finely powdered or liquid drugs that are carried into the respiratory passages by the use of special delivery systems, such as pharmaceutical aerosols, that hold a solution or suspension of the drug in a liquefied gas propellent.
- the ⁇ v ⁇ 3 integrin/VEGFR2 complex inhibiting agents may be used to treat animals and patients with aberrant angiogenesis, such as that contributing to a variety of diseases and disorders.
- arthritis rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, age-related macular degeneration, Grave's disease, vascular restenosis, including restenosis following angioplasty, arteriovenous malformations (AVM), meningioma, hemangioma and neovascular glaucoma.
- angiofibroma angiofibroma, atherosclerotic plaques, corneal graft neovascularization, hemophilic joints, hypertrophic scars, osler-weber syndrome, pyogenic granuloma retrolental fibroplasia, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis, various other inflammatory diseases and disorders, and even endometriosis.
- Further diseases and disorders that are treatable by the invention, and the unifying basis of such angiogenic disorders, are set forth below.
- angiogenesis is involved in rheumatoid arthritis, wherein the blood vessels in the synovial lining of the joints undergo angiogenesis.
- the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction.
- the factors involved in angiogenesis may actively contribute to, and help maintain, the chronically inflamed state of rheumatoid arthritis.
- Factors associated with angiogenesis also have a role in osteoarthritis, contributing to the destruction of the joint.
- ocular neovascular disease Another example of a disease mediated by angiogenesis is ocular neovascular disease. This disease is characterized by invasion of new blood vessels into the structures of the eye, such as the retina or cornea. It is the most common cause of blindness and is involved in approximately twenty eye diseases. In age-related macular degeneration, the associated visual problems are caused by an ingrowth of chorioidal capillaries through defects in Bruch's membrane with proliferation of fibrovascular tissue beneath the retinal pigment epithelium. Angiogenic damage is also associated with diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia.
- corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical bums, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginal keratolysis, rheumatoid arthritis, system ⁇ c lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, and corneal graph rejection.
- Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity;, Eales disease, Bechets disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Bests disease, myopia, optic pits, Stargarts disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications.
- Other diseases include, but are not limited to, diseases associated with rub, sarco
- Chronic inflammation also involves pathological angiogenesis.
- pathological angiogenesis Such disease states as ulcerative colitis and Crohn's disease show histological changes with the ingrowth of new blood vessels into the inflamed tissues. Bartonellosis, a bacterial infection found in South America, can result in a chronic stage that is characterized by proliferation of vascular endothelial cells.
- Another pathological role associated with angiogenesis is found in atherosclerosis. The plaques formed within the lumen of blood vessels have been shown to have angiogenic stipulatory activity. VEGF expression in human coronary atherosclerotic lesions has been demonstrated. This evidences the pathophysiological significance of VEGF in the progression of human coronary atherosclerosis, as well as in recanalization processes in obstructive coronary diseases.
- the present invention provides an effective treatment for such conditions.
- Angiogenesis is also responsible for damage found in hereditary diseases such as Osier- Weber-Rendu disease, or hereditary hemorrhagic telangiectasia. This is an inherited disease characterized by multiple small angiomas, tumors of blood or lymph vessels. The angiomas are found in the skin and mucous membranes, often accompanied by epistaxis (nosebleeds) or gastrointestinal bleeding and sometimes with pulmonary or hepatic arteriovenous fistula.
- Angiogenesis is also involved in normal physiological processes such as reproduction and wound healing. Angiogenesis is an important step in ovulation and also in implantation of the blastula after fertilization. Prevention of angiogenesis could be used to induce amenorrhea, to block ovulation or to prevent implantation by the blastula.
- Li wound healing, excessive repair or fibroplasia can be a detrimental side effect of surgical procedures and may be caused or exacerbated by angiogenesis. Adhesions are a frequent complication of surgery and lead to problems such as small bowel obstruction.
- Diseases and disorders characterized by undesirable vascular permeability can also be treated by the present invention. These include edema associated with brain tumors, ascites associated with malignancies, Meigs' syndrome, lung inflammation, nephrotic syndrome, pericardial effusion and pleural effusion, as disclosed in WO 98/16551, specifically incorporated herein by reference.
- the ⁇ v ⁇ 3 integrin/VEGFR2 complex inhibiting agents of the invention can also be utilized in the treatment of tumors.
- Tumors in which angiogenesis is important include malignant tumors, and benign tumors, such as acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.
- Angiogenesis is particularly prominent in solid tumor formation and metastasis.
- angiogenesis is also associated with blood-born tumors, such as leukemias, and various acute or chronic neoplastic diseases of the bone marrow in which unrestrained proliferation of white blood cells occurs, usually accompanied by anemia, impaired blood clotting, and enlargement of the lymph nodes, liver, and spleen.
- Angiogenesis also plays a role in the abnormalities in the bone marrow that give rise to leukemia-like tumors.
- Angiogenesis is important in two stages of tumor metastasis. In the vascularization of the primary tumor, angiogenesis allows cells to enter the blood stream and to circulate throughout the body. After tumor cells have left the primary site, and have settled into the secondary, metastasis site, angiogenesis must occur before the new tumor can grow and expand. Therefore, prevention of angiogenesis can prevent metastasis of tumors and contain the neoplastic growth at the primary site, allowing treatment by other therapeutics, particularly, therapeutic agent-targeting agent constructs.
- the ⁇ v p3 integrin/VEGFR2 complex inhibiting agents provided by this invention are thus broadly applicable to the treatment of any malignant tumor having a vascular component.
- the agents may be used alone or in combination with, e.g., chemotherapeutic, radiotherapeutic, apoptopic, anti-angiogemc agents and/or immunotoxins or coaguligands.
- Typical vascularized tumors for treatment are the solid tumors, particularly carcinomas, which require a vascular component for the provision of oxygen and nutrients.
- Exemplary solid tumors that may be treated using the invention include, but are not limited to, carcinomas of the lung, breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, squamous cell carcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas, glioblastomas, neuroblastomas, and the like.
- WO 98/45331 is also incorporated herein by reference to further exemplify the variety of tumor types that may be effectively treated using an anti-VEGF polypeptide.
- Knowledge of the role of angiogenesis in the maintenance and metastasis of tumors has led to a prognostic indicator for cancers such as breast cancer.
- the amount of neovascularization found in the primary tumor was determined by counting the microvessel density in the area of the most intense neovascularization in invasive breast carcinoma. A high level of microvessel density was found to correlate with tumor recurrence. Control of angiogenesis by the therapies of the present invention will reduce or negate the recurrence of such tumors.
- the present invention is contemplated for use in the treatment of any patient that presents with a solid tumor.
- the therapeutics of the present invention will have reduced side effects.
- Particular advantages will result in the maintenance or enhancement of host immune responses against the tumor, as mediated by macrophages, and in the lack of adverse effects on bone tissue.
- the invention will thus be the anti-angiogenic therapy of choice for the treatment of pediatric cancers and patients having, or at risk for developing, osteoporosis and other bone deficiencies.
- the unconjugated polypeptides comprising ct v ⁇ 3 integrin/VEGFR2 complex inhibiting agents are particularly contemplated for use in treating patients with more angiogenic tumors, or patients at risk for metastasis.
- the present invention is also intended as a preventative or prophylactic treatment. These aspects of the invention include the ability of the invention to treat patients presenting with a primary tumor who may have metastatic tumors, or tumor cells in the earlier stages of metastatic tumor seeding.
- the present invention may also be used to prevent tumor development in subjects at moderate or high risk for developing a tumor, as based upon prognostic tests and/or close relatives suffering from a hereditary cancer, [00194]
- Therapeutically effective doses of the ⁇ v p 3 integrin/VEGFR2 complex inhibiting agents are readily determinable using data from an animal model. Experimental animals bearing solid tumors are frequently used to optimize appropriate therapeutic doses prior to translating to a clinical environment. Such models are known to be very reliable in predicting effective anti-cancer strategies. For example, mice bearing solid tumors are widely used in pre-clinical testing.
- Any dose, or combined medicament of the ⁇ v ⁇ 3 integrin/VEGFR2 complex inhibiting agents, that results in any consistently detectable anti-angiogenic effect, inhibition of metastasis, tumor vasculature destruction, tumor thrombosis, necrosis and/or general anti-tumor effect will define a useful invention.
- the present invention may also be effective against vessels downstream of the tumor, Le., target at least a sub-set of the draining vessels, particularly as cytokines released from the tumor will be acting on these vessels, changing their antigenic profile.
- the intention of the therapeutic regimens of the present invention is generally to produce significant anti-tumor effects while still keeping the dose below the levels associated with unacceptable toxicity,
- the administration regimen can also be adapted to optimize the treatment strategy.
- Intravenous injection is generally preferred. Continuous infusion over a time period of about 1 or 2 hours or so is also contemplated.
- the present invention can be combined with other therapies.
- angiogenic diseases such as arthritis, psoriasis, atherosclerosis, diabetic retinopathy, age-related macular degeneration, Grave's disease, vascular restenosis, hemangioma and neovascular glaucoma (or other diseases described above), or solid tumors.
- the ⁇ ⁇ ⁇ 3 integrin/VEGFR2 complex inhibition based treatment methods of the present invention may be combined with any other methods generally employed in the treatment of the particular tumor, disease or disorder that the patient exhibits. So long as a particular therapeutic approach is not known to be detrimental to the patient's condition in itself, and does not significantly counteract the polypeptides comprising Oyp 3 integrin/VEGFR2 complex inhibiting based treatment, its combination with the present invention is contemplated. [00203] In accordance with another aspect of the invention, methods of, and uses in, significantly inhibiting O v p 3 integrin and VEGFR2 complex formation without inhibiting natural ligand binding to ⁇ v p 3 integrin are provided.
- These methods comprise contacting, in the presence of VEGF, a population of cells or tissues that includes a population of endothelial cells that express VEGFR2 (KDR/Flk-1) and ⁇ v p 3 integral with a composition comprising a biologically effective amount of at least one agent that inhibits tyrosine phosphorylation of a v p 3 integrin and/or VEGFR2.
- Proliferation inhibition methods and uses are provided, including those to specifically inhibit VEGF-induced endothelial cell proliferation and/or migration, which generally comprise contacting a population of cells or tissues that includes a population of endothelial cells and VEGF with a composition comprising a biologically effective amount of the at least one ⁇ v p 3 integrin/VEGFR2 complex inhibiting agent under conditions effective to inhibit VEGF-induced endothelial cell proliferation and/or migration.
- the foregoing methods and uses can be performed in vitro and in vivo. In the latter case the tissues or cells are located within an animal and the at least one ⁇ v p 3 integrin/VEGFR2 complex inhibiting agent is administered to the animal.
- the methods and uses become methods and uses for inhibiting angiogenesis, comprising contacting a tissue comprising, or a population of, potentially angiogenic blood vessels, Le,, those potentially exposed to VEGF, with an anti-angiogenic composition comprising a biologically effective amount of the at least ⁇ v ⁇ 3 integrin/VEGFR2 complex inhibiting agent under conditions effective to inhibit angiogenesis.
- the present invention has utility in drug discovery programs.
- In vitro screening assays with reliable positive and negative controls, are useful as a first step in the development of drugs to inhibit or promote angiogenesis, as well as in the delineation of further information on the angiogenic process.
- the anti-angiogenic composition is administered to the animal as a form of therapy.
- Bioly effective amounts in terms of each of the foregoing inhibitory methods are therefore amounts of the at least one 0 ⁇ 3 integrin/VEGFR2 complex inhibiting agent effective to inhibit ⁇ v p 3 integrin and VEGFR2 complex formation without substantially inhibiting natural or native ligand binding to ⁇ ⁇ ⁇ 3 integrin.
- the present invention thus further provides methods of, and uses in, treating a disease associated with angiogenesis, including all forms of cancer associated with angiogenesis, comprising administering to an animal or patient with such a disease or cancer a therapeutically effective amount of at least a first pharmaceutical composition that comprises at least O v p 3 integrin/VEGFR2 complex inhibiting agent.
- the foregoing anti-angiogenic treatment methods and uses will generally involve the administration of the pharmaceutically effective composition to the animal or patient systemically, such as by transdermal, intramuscular, intravenous injection and the like.
- any route of administration that allows the therapeutic agent to localize to the angiogenic site or sites including tumor or intratumoral vascular endothelial cells, will be acceptable. Therefore, other suitable routes of delivery include oral, rectal, nasal, topical, and vaginal.
- U.S. Pat. No. 5,712,291 is specifically incorporated herein by reference for purposes including further describing the various routes of administration that may be included in connection with the treatment of an angiogenic disease or disorder.
- ophthalmic formulations and administration are contemplated.
- administering means provision or delivery of the Ov ⁇ 3 integrin/VEGFR2 complex inhibiting agent in an amount(s) and for a period of time(s) effective to exert anti-angiogenic and/or anti-tumor effects.
- the passive administration of proteinaceous therapeutics is generally preferred, in part, for its simplicity and reproducibility.
- Integrin phosphorylation and signaling is critical for
- Tyrosine phosphorylation of ⁇ s cytoplasmic motif in EC occurs upon adhesion to integrin Iigands.
- Extracellular matrix proteins which serve as Iigands for ⁇ v ⁇ 3 such as vitronectin, f ⁇ bronectin, fibrinogen and gelatin strongly stimulated Tyr759 and Tyr747 phosphorylation.
- Sodium pervanadate known to block phosphatase activity was used as a positive control in this experiment. As anticipated, no tyrosine phosphorylation of ⁇ 3 integrin was observed in DiYF EC under any conditions.
- VEGF treatment of EC induces tyrosine phosphorylation of beta 3.
- ⁇ 3 subunit phosphorylation in extracellular matrix recognition was previously assessed in model cell lines, such as CHO cells and K562 cells. This system does not provide any insight on the most important functions of the ⁇ 3 integrin, namely its role in angiogenesis and in the regulation of VEGF or FGF-induced EC responses. These processes can be only assessed in EC expressing appropriate receptors and signaling intermediates. Thus, we determined whether VEGF treatment is able to affect the phosphorylation status of ⁇ 3 . Treatment of EC in suspension with VEGF-Al 65 induced Tyr747 as well as Tyr 759 phosphorylation of ⁇ 3 in a time-dependent manner.
- Angiogenesis in vivo is impaired in DiYF mice.
- VEGF-induced angiogenesis was significantly impaired in DiYF mice.
- mice were assessed tumor-induced pathological angiogenesis in DiYF and WT mice.
- mouse melanoma cells were implanted subcutaneously into mice and 10 day after, tumors were excised.
- Tissue section analysis revealed the presence of well-developed blood vessels which were positively stained for vWF, CD31 and Iaminin (basement membrane component) in tumors from WT mice.
- Fig. 3B blood vessels were sparse and thin-walled based on laminin staining
- Tyrosine phosphorylation of ⁇ 3 integrin controls ex vivo angiogenesis in response to VEGF.
- aortic ring sprouts revealed the ability of DiYF cells to form vascular sprouts ex vivo was at least 4 fold lower regardless of stimulation (Fig. 4G).
- VEGF produced a mild increase in capillary formation of DiYF rings, however, the number of sprouts was only 20-25% of that in WT aortic rings (Fig. 4G).
- a detailed kinetic study was undertaken. The time curves of vascular growth are presented in Fig. 4H.
- WT and DiYF EC were plated on various integrin ligands and were allowed to form a confluent monolayer. Then, a wound in the monolayer was created and the healing process was monitored at different time points.
- the quantitative aspects of wound recovery and representative images of EC are presented in Figs. 5E and 5F, respectively. Where as WT and DiYF EC migrated equally well on fibronectin, laminin and collagen, a 3 fold reduction in migration on vitronectin was observed in DiYF EC compared to WT (Fig. 5E). Moreover, using live video microscopy, the process of EC migration and wound recovery was carefully monitored in order to characterize differences in cell movement between WT and DiYF EC (See supplemental data).
- DiYF mutation impairs ⁇ v p 3 integrin-dependent and VEGF-stimulated responses of EC indicating its potential role in the regulation of a cross-talk between ⁇ v ⁇ 3 and VEGF receptor(s).
- p 3 integrin phosphorylation is required for sustained activation of VEGF receptor-2.
- VEGFR-2 phosphorylation status in response to VEGF in WT and DiYF EC.
- a time course of VEGFR2 phosphorylation is presented in Fig. 6C. Ih WT EC, VEGF induced a bell-shaped response with a maximum 6 fold increase in VEGFR-2 phosphorylation over control. After 45 min, VEGFR-2 phosphorylation returned to the control levels. In contrast, VEGF exerted much lower increase in VEGFR-2 phosphorylation in DiYF EC with the maximum value of 2.5 fold over control.
- VEGFR-2 in WT EC remained phosphorylated 3 times longer than in DiYF EC (Fig. 6C).
- the lack of integrin phosphorylation in DiYF EC resulted in reduced phosphorylation/activation of VEGFR-2 in response to VEGF, which, in turn, affected all the signaling events downstream of VEGFR-2.
- Tyrosine phosphorylation is critical for VEGF-induced oufo integrin activation.
- integrin activation An intrinsic property of integrins is an increased soluble ligand binding in response to stimulation, a process referred to as integrin activation.
- VEGF via VEGFR-2 is able to activate ⁇ v ⁇ 3 integrin on EC. Accordingly, we sought to determine whether impaired activation of VEGFR-2 in DiYF EC results in defective ⁇ v ⁇ 3 activation by VEGF.
- VEGF induced at least 6 fold increase of fibrinogen binding to WT EC and only 3 fold increase of binding to DiYF EC (Fig. 6D).
- MnCl 2 an agonist known to activate integrins and at the same time to stimulate ⁇ 3 integrin tyrosine phosphorylation, , produced at least 40 fold increase in fibrinogen binding to WT EC, compared to 17 fold increase observed in DiYF EC (Fig. 6E).
- the specificity of ligand binding was conformed by addition of 10 fold excess of unlabelled fibrinogen. Similar results were observed when integrin activation was monitored using a monovalent activation-dependent ligand WOW-I Fab.
- VEGF and MnCl 2 stimulated 9 and 30 fold increases, respectively, in WOW-I binding to WT EC and 3.5 and 14 fold increases, respectively, to DiYF EC (Fig. 6F and 6G).
- DiYF mutations within the cytoplasmic domain of ⁇ 3 integrin significantly impair the process of integrin activation, which, in turn, results in defective cell adhesion and migration.
- DiYF EC ⁇ 3 integrin is present on the surface at the normal level; however, its function has been modified by the mutations.
- the second unique aspect of our experimental system is the lack of compensatory or over- compensatory responses, which are often observed in knockout models when the protein of interest, is absent during development.
- the knockin technique seems to be more physiologically relevant and more direct compared to the knockout approach.
- this particular technique is the most appropriate for the multifunctional proteins involved in various signaling pathways since it allows focusing on the certain function of the protein, in our case on the role of tyrosine phosphorylation without disturbing other aspects of ⁇ 3 integrin activity.
- DiYF knockin mice we assessed angiogenesis in vivo as well as performed an extensive analysis of the underlying mechanisms using numerous ex vivo models and the major findings of this manuscript are the following: 1) Phosphorylation of p 3 integrin in WT EC occurs in response to integrin ligation as well as VEGF stimulation; 2) Lack of ⁇ 3 integrin phosphorylation in DiYF knockin mice results in impaired angiogenic response and reduced tumor growth in vivo; 3) VEGF-induced functional responses of EC from DiYF mice (cell adhesion, spreading, migration and capillary tube formation) are defective compared to WT; 4) Lack of ⁇ 3 integrin phosphorylation in DiYF knockin EC leads to disruption of VEGFR-2/ ⁇ 3 integrin complex formation and causes a deficiency of VEGFR-2 phosphorylation in response to VEGF; 5) VEGF-induced integrin activation (inside-out signaling) is suppressed in DiY
- DiYF mice were generated in the laboratory of Dr. David R. Phillips and maintained on C57/B16 background (7 generations of backcrossing). Six to eight week old wild-type (WT) and DiYF mice were used in study.
- Wild type and DiYF mouse lungs were excised, minced and digested using collagenase-dispase reagent (3 mg/ml). Digests were strained and the resulting cell suspension was plated on flasks coated with 1 mg/ml fibronectin. Endothelial cells were isolated and characterized.
- Thoracic aortas from WT and DiYF mice were removed under aseptic conditions and spliced into lmm thick rings. Thoracic aortic rings were placed between two layers of growth factor depleted Matrigel and allowed to solidify at room temperature. Matrigels were overlaid with either with DMEM or endothelial growth medium with or without VEGF (40ng/ml). Microvessel outgrowth was visualized by phase contrast microscopy and numbers of vessels growing from each aortic ring were counted and photographed every two days using Leica phase contrast microscope.
- Mouse lung endothelial cells were detached from the tissue culture flasks using 20 mM EDTA. Cells were washed twice with sterile PBS and re- suspended in DMEM. The cell suspensions were added to ligand-coated wells and placed in humidified incubator for 45 min. The wells were gently washed three times with DMEM and photographs were taken. The numbers of attached and spread cells per field were counted.
- Transwell tissue culture inserts were coated with various integrin ligands for 24h at 4 0 C. Both WT and DiYF lung endothelial cells were trypsi ⁇ ized and IXlO 5 cells were added into each well. The lower chamber contained varying concentrations of VEGF-A 165 (0-20 ng/ml). Cells were allowed to migrate for 12 h and fixed with 3.7% formaldehyde/PBS for 15 min and stained with 0.5% crystal violet. The non-migrated cells adhered to the top surface were removed and three random 1OX fields were photographed using Leica inverted phase contrast microscope.
- WT and DiYF mouse lung endothelial cells were grown to confluence in 12 well plates precoated with various integrin ligands. Cells were serum starved for 4h and then a wound was created by a 1000 ⁇ l pipette tip. Wells were rinsed twice with sterile PBS to remove wound-derived loose and dislodged cells and further cultured DMEM medium containing 2% FBS. Images were recorded immediately after wounding (time zero) and 12 h later. Cell migration was quantified using image analysis of 5 randomly selected fields of denuded area.
- Wild type and DiYF mouse lung endothelial cells were serum starved for 4h and further stimulated with 20 ng/ml VEGF-165, 20 ng/ml b-FGF or Imm MnCL2 separately.
- WoW-I Fab was added at a final concentration of 30 mg/ml, followed by addition of Fluorescein isothiocyanate (FITC) conjugated goat anti- mouse IgG at 10 ⁇ g/ml. After 30 min cells were fixed with 3.7% formaldehyde/PBS for 15 min, washed twice with IX PBS and FACS was performed using a FACS Calibur (Becton Dickinson, San Jose, CA) instrument and data were analyzed using CellQuest software program.
- FITC Fluorescein isothiocyanate
- Wild type and DiYF mouse lung endothelial cells were trypsinized and washed twice with DMEM containing 10% FCS. These cells were seeded on Matrigel coated 6 well plate and cells were allowed to adhere. After 24h, the medium was removed and cells were overlaid with 0.5 ml of Matrigel containing 40 ng/ml VEGF. All the wells were filled with 2 ml of endothelial growth medium and cells were observed and photographed every day using Leica phase contrast microscope.
- Integrin immune complexes from agarose beads samples were boiled for 5 min in SDS-PAGE sample buffer. Immunocomplexes were resolved by SDS- PAGE (6%), and transferred to nitrocellulose membrane. These blots were probed with anti- VEGFR-2 and anti-b3 integrin antibody respectively. Proteins were detected using enhanced chemiluminescence technique (Amersham).
- Cell lysates were analyzed by Western blot using anti-integrin ⁇ 3 [pY 7 7 ], anti-integrin [pY 5 ], anti-p-VEGFR-2, anti- VEGFR-2, anti-p-ERKl/2, anti-ERKl/2, anti-p-Akt, anti-Akt, anti-p-P38MAPK and anti-MAPK antibody.
- the wild type and DiYF endothelial cells were lysed and equal proteins from total cell lysates were subjected SDS-PAGS and analyzed by western blot using anti-VEGFR-2 and anti-CD-31 antibody.
- the aim of this study was to assess the role of each individual subfamily of integrin receptors in VEGF -induced angiogenic cellular responses using a siRNA-based short-term knockdown approach in primary endothelial cells.
- Rabbit polyclonal anti-VEGFR-2, anti- ⁇ 3-integrin, anti- ⁇ 5-integrin, anti ⁇ l -integrin, mouse monoclonal anti-phospho tyrosine (PY20 and PY99) antibodies, and ⁇ l, ⁇ 3, and ⁇ 5 integrin-specific siRNAs were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
- Anti-VEGFR-2 and anti-phospho VEGFR-2 were from Cell Signaling Technology (Beverly, MA).
- Mouse monoclonal anti- ⁇ 3-integrin blocking antibody was from Chemicon International Inc. (Temecula, CA).
- Anti-CD31 antibody was obtained from DAKO (Kyoto, Japan).
- HUVEC Human umbilical cord vein endothelial cells
- HUVECs were detached from the tissue culture flasks using 20 mM EDTA. Cells were washed twice with sterile phosphate buffered saline (PBS) and re-suspended in serum-free DMEN. The cell suspensions were added to integrin ligand-coated wells and placed in a humidified incubator for 45 min. The wells were gently washed three times with DMEM and photographs were taken. The numbers of attached and spread cells per field were counted.
- PBS sterile phosphate buffered saline
- HUVECs were grown to confluence in 12 well plates precoated with various integrin ligands. Cells were serum starved for 4 h and then a wound was created by a pipette tip. Wells were rinsed twice with sterile PBS to remove wound-derived loose and dislodged cells and further cultured in DMEM medium containing 2% FBS. Images were recorded immediately after wounding (time zero) and 12 h later. Cell migration was quantified using image analysis of five randomly selected fields of denuded area.
- HUVECs Sem ⁇ confluent HUVECs were serum starved for 4 h and further stimulated with 20 ng/mL VEGF-A165 or VEGFD ⁇ N ⁇ C WOW-I Fab was added to a final concentration of 30 ⁇ g/mL, followed by addition of FITC- conjugated goat anti-mouse IgG at 10 ⁇ g/mL. After 30 min cells were fixed with 3.7% formaldehyde in PBS for 15 min, washed twice with PBS, and fluorescence- activated cell sorting (FACS) was performed using a FACS Calibur (Becton Dickinson, San Jose, CA) and data were analyzed using CellQuest software. Tube formation assay
- HUVECs The formation of vascular tube-like structures by HUVECs was assessed on a basement membrane matrix preparation. Twelve-well plates were coated with 0.5 mL of Matrigel according to the manufacturer's instructions. HUVECs transfected with various ⁇ integrin-specific siRNAs. Cells were detached from tissue culture flasks using 20 mM EDTA in PBS. Cells were washed twice with sterile PBS and seeded on Matrigel-coated plates. Medium with or without 20 ng/mL VEGF was added and cells were further incubated at 37 0 C for 8 h. The tube formation was observed using an inverted phase contrast microscope (Leica, Wetzlar, Germany) and photographs were taken. Using ImagePro software (Media Cybernetics, Silver Spring, MD), the degree of tube formation was quantified by measuring the length of tubes in three random fields.
- ImagePro software Media Cybernetics, Silver Spring, MD
- HUVECs were grown in monolayer on glass slides and then treated with 1 mM MnCl 2 or 20 ng/mL VEGF for 10 min. These cells were further incubated with WOW-I antibody for additional 30 min. These cells were fixed with paraformaldehyde for 10 min, blocked with 5% bovine serum albumin for 30 min, and incubated with FITC-conjugated anti-mouse IgG. These cells were washed, mounted with coverslips, and analyzed under a florescent microscope (Leica). Alternatively, cells were further incubated with anti- VEGFR- 2 antibody and incubated with TRITC-conjugated anti-rabbit IgG. These cells were washed, mounted, and analyzed under confocal microscopy (Leica).
- HUVECs were lysed following the experiment using lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 10 mM NaF, 10 mM Na2P2O7, 10 ⁇ g/mL leupeptin, 4 ⁇ g/mL pepstatin, and 0.1 units/mL aprotinin).
- lysis buffer 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM iodoacetamide, 2 mM phenylmethylsulfonyl flu
- Cell lysates were cenrrifuged at 13,000 x g for 10 min at 4 0 C. Supernatants were collected and assayed for protein concentration using the Bio-Rad protein assay method (Hercules, CA). Cell lysates were denatured using Laemmli sample buffer and proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and probed with indicated antibody. When appropriate, nitrocellulose membranes were stripped and blotted according to manufacturer's instructions.
- siRNAs small interfering RNAs
- ECM extracellular matrix
- integrin ligand ligation induces a wide variety of intracellular signaling, including tyrosine phosphorylation of FAK, increased inositol lipid synthesis, cyclin synthesis, and expression of several cell survival factors.
- Endothelial cell surface integrins mediate adhesion to ECM proteins, including vitronectin, laminin, collagen, von Willebrand factor, and fibrinogen.
- HUVECs transfected with integrin- specific siRNA were plated on various integrin ligand coated plates and ability of EC to adhere to the distinct extracellular matrix components were tested. Representative images and quantitative results are shown in Fig. &4 and %B, respectively.
- Inhibition of expression of the ⁇ l subunit abolished EC adhesion to both collagen and laminin, but not to vitronectin.
- Silencing of the ⁇ 3 subunit completely inhibited EC adhesion to vitronectin, but not to collagen or laminin.
- Vitronectin ( ⁇ v ⁇ 3) and collagen ( ⁇ 5 ⁇ l) receptors modulate endothelial cell migration.
- Extracellular matrix provides critical support for proliferating vascular endothelium through adhesive interaction with endothelial cell surface integrins. Extracellular matrix also provides the scaffold essential for maintaining the organization of vascular endothelial cells in to blood vessels. Endothelial cell adhesion to extracellular matrix is required for endothelial cell proliferation, migration, and morphogenesis. Integrin-mediated migration of endothelial cells also plays a crucial role in vascular remodeling involved in angiogenesis, embryonic vasculogenesis, and re-endothelialization in arteries following angioplasty.
- HUVECs were transfected with various ⁇ -integrin specific siRNAs and these cells were plated on various extracellular matrix components.
- a wound was created across the cell monolayer by scraping away a swath of cells and the extent of wound healing due to the transfected EC was measured after 12 hours. Representative images are shown in Fig. 9A. Percentages of wound recovery were quantified (Fig. 9B). Control EC were able to completely close the wound on vitronectin and was assigned value of 100%.
- EC plated on collagen and laminin shown 85% and 30% recovery respectively.
- endothelial cells organize to form three-dimensional capillary networks. This processes initiates with transition of endothelial cells into spindle-shaped morphology. This is followed by endothelium alignment and connection into solid, multicellular, precapillary cord-like structures that form an integrated polygonal network.
- extracellular matrix serves as an adhesive support and, through interaction with integrins, provides crucial signaling to regulate endothelial cell shape and contractility.
- HUVECs were transfected with various ⁇ -integrin specific siRNAs and angiogenic properties of EC were assessed using a capillary tube formation assay on Matrigel. Capillary tube forming ability of unstimulated as well as VEGF-stimulated cells was tested. Silencing of the ⁇ l subunit affected capillary formation in the absence as well as in the presence of VEGF. As shown in Fig. IQi, the regularity of the typical honeycomb-like pattern was disturbed resulting in incomplete connections between cellular cords.
- Integrin affinity relates to conformational modification in the integrin heterodimer to strength of ligand binding.
- non-integrin receptors cause alterations in the integrin cytoplasmic domain ultimately modulating integrin activation state.
- RTKs receptor tyrosine kinases
- G-protein coupled receptors G-protein coupled receptors
- cytokines have been shown to modulate integrin activation state.
- HUVECs were induced with VEGF and integrin affinity was estimated.
- a genetically engineered antibody WOW-I was used as a probe to detect ⁇ v ⁇ 3 in the high affinity state on the surface of EC in the monolayer.
- WOW-I Fab is a monomeric soluble ligand which binds only to the activated form of ⁇ v p 3 .
- Stimulation with VEGF-AI65 increased WOW-I binding to EC by 6-fold compared to resting cells as measured by FACS analysis (Fig. 11,4).
- VEGF-D vascular endothelial growth factor-D
- VEGF-D ⁇ N ⁇ C vascular endothelial growth factor-2
- WOW-I Fab binding indicating that VEGFR-2 but not VEGFR-I is primarily receptor mediating ⁇ v ⁇ 3 integrin activation.
- Activated ⁇ V ⁇ 3 integrin is index for enhanced tumor vasculature.
- VEGFR-2/B3 integrin cross-talk in EC VEGFR-2/B3 integrin cross-talk in EC.
- Endothelial cells express a relatively wide range of integrin receptors, which permits interactions with numerous extracellular matrix ligands. Although the ligand recognition profile of each integrin is rather unique, there is a significant degree of redundancy and functional overlap. This is a case for ⁇ v ⁇ 3 and ⁇ v ⁇ s, since both integrins are considered as primary receptors for vitronectin. However, our data indicate that of the several integrin receptors on EC, including 0 ⁇ 3 has a distinct function in angiogenesis, which cannot be compensated by other mtegrins.
- affinity of ⁇ v ⁇ 3 on EC can be rapidly modulated by the treatment with VEGF, resulting in increased adhesive and migratory responses of EC.
- ⁇ v p 3 is activated in endothelium of highly vascularized tumor as compared to normal tissues.
- the pro-angiogenic role of ⁇ v ⁇ 3 integrin is not limited to interactions with vitronectin and other substrates; it is tightly linked to its ability to augment activation of VEGFR-2.
- activated ⁇ v ⁇ 3 is co-localized with VEGFR-2 not only in cultured EC, but also on endothelial cells of proliferating blood vessels at the sites of pathological angiogenesis.
- siRNA-based knockdown approach used in our study and genetic ablation in knockout animals is centered on the transient nature of siRNA- based silencing versus permanent and complete loss of protein expression in development If the function of targeted protein cannot be spared during development, such loss will ultimately result in embryonic lethality.
- VEGF does not seem to be absolutely required for normal physiology of adult organisms, since neutralization of circulating VEGF has proven safe in patients.
- this integrin is not likely to be a crucial player during development but it does mediate pathological neovascularization in adults.
- Method angiogenesis assay in matrigel.
- ⁇ v p 3 /VEGFR2 complex inhibiting peptides in accordance with the present invention was assessed by tube formation assay on the basement membrane matrix preparation.
- Six well plates were coated with 0.5 mL of Matrigel according to the manufacturer's instructions.
- HUVECs Human Umbilical vascular endothelial cells
- Cells were washed twice with sterile PBS and seeded on Matrigel-coated plates in combination of presence/ absence of VEGF and inhibitory peptide. Cells were further incubated at 37 C for 8 h.
- Fig. 14 illustrates (A) photographs of endothelial cell tube formation in a matrigel assay for endothelials cells subjected to VEGF, a peptide in accordance with the present invention, and VEGF in conjunction with a peptide in accordance with the present invention; and (B) a graph showing the results.
- HUVEC cells were serum starved for 3 hours and further incubated with these antibodies for 2 hours at room temperature. Cells were induced with 20ng/ml VEGF for 5 mins and analyzed for phosphorylation of beta3 integrin by Western blot analysis.
- Example 4
- Matrigel plug assays were performed in age- and sex-matched C57/B16 background mice. Each animal received an abdominal subcutaneous injection of 500 ⁇ L Matrigel mixed with VEGF (60 ng/mL), heparin (60 units/mL) and 50 mM of control (YGRKKRRORRR G DTANNPL FKEATSTFT- COOH) (SEQ ID NO: 14) or beta-3 integrin cytoplasmic domain inhibitory peptide (YGRKKKRQRRR G DTANNPL Yp KEATSTFT- COOH) (SEQ ID NO: 2). After 7 days, the animals were euthanized and dissected. Matrigel plugs were removed and digested using 5 mL Drabkin reagent and neovascularization was assessed using a hemoglobin assay as per the manufacturer's protocol (Fig. 16).
- vitronectin but not laminin or collagen was able to induce ⁇ 3 integrin tyrosine phosphorylation, which was augmented upon VEGF treatment.
- phosphorylation of ⁇ 3 integrin was minimal in cells in suspension or plated on laminin or collagen despite the stimulation with VEGF (Fig. 18A).
- VEGFR-2 phosphorylation at Tyr-1175 in the same set of samples revealed that basal level of VEGFR-2 activation can be triggered by ⁇ v ⁇ 3 integrin ligation by vitronectin but does not occur in cells plated on ⁇ 2 ⁇ l integrin ligand (collagen) or ⁇ 6 ⁇ l/ ⁇ 6 ⁇ 4 integrin ligand (laminin).
- ⁇ v ⁇ 3 integrin ligation by ⁇ v ⁇ 3 also stimulates VEGFR-2 phosphorylation and activation demonstrating a mutual relationship between VEGFR2 and ⁇ v ⁇ 3.
- ⁇ 3 integrin tyrosine phosphorylation is complementary for VEGF induced tyrosine phosphorylation of VEGFR-2.
- ⁇ v ⁇ 3 integrin is expressed on proliferating endothelial cells during angiogenesis and vascular remodeling and the blockade of ⁇ v ⁇ 3 integrin suppressed angiogenesis in several in vivo models. Therefore, we assessed whether the blockade of ⁇ v ⁇ 3 affected tyrosine phosphorylation of ⁇ 3 subunit. Accordingly, HUVECs grown on gelatin coated plates were serum starved and further incubated with 10 ⁇ g/mL anti- ⁇ v, anti- ⁇ 3, anti- ⁇ l, anti- ⁇ 5 blocking antibodies at 4°C for 2 hours. Cells were also treated with non-immune IgG as control.
- ⁇ 3 integrin Tyr-747 and 759
- Fig. 18B shows that both anti- ⁇ v and anti- ⁇ 3 blocking antibodies inhibited VEGF induced tyrosine phosphorylation of ⁇ 3 integrin at both tyrosine- 747 and tyrosine759.
- control IgG, anti- ⁇ l or anti- ⁇ 5 blocking antibody did not have substantial effect on VEGF -induced tyrosine phosphorylation of ⁇ 3 integrin.
- VEGFR-2 inhibition significantly reduced VEGF induced ⁇ 3 integrin cytoplasmic tyrosine residue phosphorylation (Fig. 18 panel D).
- ⁇ v ⁇ 3 integrin ligation controls not only tyrosine phosphorylation of the ⁇ 3 subunit but also of VEGFR-2
- VEGF stimulation promotes not only VEGFR2 but also ⁇ 3 integrin tyrosine phosphorylation.
- VEGFR-2 activation and signaling seems to be tightly associated with integrins, we intend to examine interaction of VEGFR-2 with various integrin on endothelial cell surface.
- VEGF differentially induces interaction of Src family tyrosine kinases (SFKs) with B3 integrin in endothelial cells
- ⁇ subunit of integrin heterodimers are known to associate with Src family tyrosine kinases (SFKs) and are required for early consequences of integrin signaling. Presence of SFKs is critical for the tyrosine phosphorylation of several intracellular signaling molecules following the integrin mediated cell adhesion and cell spreading. Therefore, we sought to analyze the degree of different SFKs interaction with ⁇ 3 integrin and VEGFR-2 upon stimulation of endothelial cells with VEGF.
- Src family tyrosine kinases SFKs
- Presence of SFKs is critical for the tyrosine phosphorylation of several intracellular signaling molecules following the integrin mediated cell adhesion and cell spreading. Therefore, we sought to analyze the degree of different SFKs interaction with ⁇ 3 integrin and VEGFR-2 upon stimulation of endothelial cells with VEGF.
- HUVECs were induced with VEGF for 10 min and cell lysates were split in to two parts and immunoprecipitated with anti- ⁇ 3 integrin and anti-VEGFR-2 antibody separately using ProFound co-immunoprecipitation kit.
- VEGF stimulation dramatically enhanced interaction of c-Src with VEGFR-2 and ⁇ 3 integrin. No basal level or VEGF induced interaction was found between Fyn and ⁇ 3 integrin or VEGFR-2.
- VEGF induced tyrosine phosphorylation of ⁇ 3 integrin at 2.5 min and remain phosphorylated up to 30 min. Maximum level of ⁇ 3 integrin tyrosine phosphorylation was observed between 5 min to 15 min. VEGF also induced similar pattern of activation phosphorylation of c-Src in endothelial cells (Fig. 19 panel C). These observations let us consider activated c-Src might be directly or indirectly responsible for ⁇ 3 integrin phosphorylation, which, in turn, is crucial for activation of ⁇ 3 integrin dependent cellular signaling and endothelial cell functions.
- Adhesion and growth factor induced ⁇ 3 integrin tyrosine phosphorylation is mediated through c-Src.
- Cytoplasmic domains of integrin ⁇ subunits are highly conserved and represents region of structural homology between the various groups.
- Cytoplasmic tail consists of a membrane proximal sequence that forms an amphipathic helix and distal well conserved sequences, NPXY and NXXY that comprise tight beta turns. These regions have been linked to the ability of integrin to localize to focal contacts, FAK phosphorylation, ligand binding affinity, integrin dependent actin cytoskeletal reorganization, cellular adhesion and spreading.
- HUVECs cells were either kept in suspension (Fig. 20, panel A, lane-1) or plated on vitronectin (lane-2) and then treated with VEGF (lane-3) or c-Src inhibitor SU6656 (lane-4). Cell lysates were analyzed for the phosphorylation of ⁇ 3 integrin. As anticipated, in comparison to HUVEC kept in suspension, cells plated on vitronectin showed high level of ⁇ 3 integrin tyrosine phosphorylation at Tyr-747 and Tyr-759 which was further augmented by VEGF.
- HUVEC HUVEC were transfected with wild type (WT Src), dominant negative (DN Src) and catalytically active (CA Src) forms of Src.
- WT Src wild type
- DN Src dominant negative
- CA Src catalytically active
- SYF+ c-Src cell line was generated by reintroducing wild type c-Src in SYF cells using retroviral vector pLXSH, As shown in Fig. 20B, no ⁇ 3 integrin tyrosine phosphorylation was observed in any of these cell types plated on uncoated plastic surface or kept in suspension (lane 2 and 3). Attachment to vitronectin stimulated high level of ⁇ 3 integrin tyrosine phosphorylation in Src-H- and SYF -f- c-Src cells but not in SYF cells (lane 4). Cells plated on laminin or collagen shown very low ⁇ 3 integrin tyrosine phosphorylation (lane 5).
- c-Src controls cell adhesion as well as VEGF induced ⁇ 3 integrin tyrosine phosphorylation.
- c-Src directly phosphorylates cytoplasmic tyrosine motifs of 03 integrin.
- Immunocomplex were incubated with a full-length purified ⁇ 3 integrin cytoplasmic domain, and [ ⁇ - 32 P] ATP incorporation into was monitored. As shown in Fig. 20D 5 the immunoprecipitated c-Src can phosphorylate ⁇ 3 integrin cytoplasmic domain (lane 2). The specific inhibitor of c-Src SU6656 blocked this process, confirming the specificity of the reaction (lane 3). Recombinant protein tyrosine phosphatase also prevented phosphorylation, indicating that it is a tyrosine substrate that is being phosphorylated (lane 4).
- c-Src activity was modified by over expression of wild type, dominant negative or catalytically active form of Src or by treatment of cells with Src and VEGFR-2 inhibitors and then, interaction between VEGFR2 and ⁇ 3 integrin was assessed.
- non-stimulated cells no interaction between ⁇ 3 integrin and VEGFR-2 was observed (Fig. 21 A and B 5 Lanel), whereas VEGF induced strong interaction between VEGFR-2 and ⁇ 3 integrin (lane-2).
- ⁇ 3 integrin cytoplasmic tyrosine motifs in ⁇ 3 integrin and VEGFR-2 interaction we utilized lung EC derived from a ⁇ 3 knock-in mouse in which Tyr747 and Tyr759 were mutated phenylalanines (DiYF).
- the cytoplasmic domain of DiYF integrin is unable to undergo phosphorylation, resulting in deficient integrin signaling.
- Fig. 21 C shows that VEGF induced phosphorylation of ⁇ 3 integrin at Tyr-747 and Tyr-759 in wild type but not in DiYF EC.
- integrin activation An intrinsic property of integrin is an increase in soluble ligand binding in response to stimulation, a process referred to as integrin activation.
- integrin activation a process referred to as integrin activation.
- HUVECs were transfected with wild type, dominant negative and catalytically active form of c-Src and stimulated with VEGF, and ⁇ v ⁇ 3 activation was assessed by WOW-I binding as described in methods. As anticipated, VEGF induced 6-fold increase in WOW-I binding (Fig. 21E).
- c-Src and c Src-dependent ⁇ 3 integrin cytoplasmic tyrosine motifs phosphorylation is essential for VEGF induced ⁇ v ⁇ 3 integrin activation as well as ligand binding to activated integrin, which are crucial steps in integrin signaling.
- c-Src required for ⁇ v ⁇ 3 integrin dependent cellular adhesion to distinct ligand.
- DiYF endothelial cells shown significant impairment in adhesion on both vitronectin as well as bone sialoprotein (Fig. 22 D). No significance differences in adhesion were found between wild type and DiYF endothelial cells suspended on BSA coated plates. These results clearly indicated that ⁇ 3 integrin cytoplasamic tyrosine motifs and c-Src mediated phosphorylation of these tyrosine residues are crucial for ⁇ v ⁇ 3 integrin dependent cellular adhesion to distinct extracellular matrix ligands. c-Src dependent phosphorylation of ⁇ 3 integrin cytoplasmic tyrosine motifs are required for ⁇ v ⁇ 3 integrin dependent directional migration of endothelial cells. : ]
- EC motility is the defining feature of angiogenesis required for the organization of proliferating EC into vessel like structures. This process requires tight regulation of interactions between cells and surrounding ECM.
- HUVECs transfected with various forms of Src were evaluated in migration assays using VEGF as an agonist and vitronectin as a substrate.
- VEGF vascular endothelial growth factor
- CA-Src constitutively active form of Src
- c-Src Pharmacological inhibitors of c-Src (SU6656) and VEGFR-2 (SU1498), which inhibit ⁇ 3 integrin tyrosine phosphorylation, also reduced EC migration triggered by VEGF (Fig. 23 A).
- c-Src mediated ⁇ 3 integrin cytoplasmic tyrosine motifs phosphorylation in ⁇ v ⁇ 3 integrin dependent endothelial cell migration
- wild type and DiYF mouse lung microvascular endothelial cells were transfected with wild type, dominant negative and catalytically active form of c- Src and stimulated with VEGF (Fig. 23 B).
- VEGF stimulation induced 2.5 fold increase in WT-Src transfected and 3.5 fold increase in CA-Src transfected wild type endothelial cell migration.
- DN-Src significantly impaired VEGF induced endothelial cell migration only in wild type endothelial cells.
- c-Src activity modification did not show any significant difference in migration of DiYF endothelial cells.
- wild type and DiYF EC also shown similar migration rate of 43 + 3.5 ⁇ m/hour and 44 + 3.3 ⁇ m/hour, respectively.
- wild type and DiYF EC showed relatively slow but similar migration at 30 + 3.2 ⁇ m/hour and 28 + 2.7 ⁇ m/hour, respectively.
- random movement of cells was distinguished from directed migration, the results became quite different.
- the average distance of directed migration from site of origin was quite low for wild type cells on laminin and collagen, 71 + 6.2 and 92 + 7.2 ⁇ m respectively (Fig. 23C). No differences between wild type and DiYF cells were found.
- wild type endothelial cells showed maximum directed migration when plated on vitronectin (229 + 6.6 ⁇ m).
- directed migration in DiYF cells were dramatically impaired (77 ⁇ 5.4 ⁇ m vs 229 +6.6 ⁇ m for wild type).
- Wild type EC plated on ⁇ v ⁇ 3 mtegrin ligand vitronectin following induction with VEGF-A 165 resulted in higher directional persistence of cell migration. Even though VEGF induced the migration rates in the cells plated on laminin and collagen, but VEGF effects were only evident in the wild type cells plated on vitronectin.
- Either VEGF or various form of Src fails to modify degree of tube formation in DiYF endothelial cells. From all these results we concluded that c-Src dependent ⁇ 3 integrin cytoplasmic tyrosine motifs phosphorylation is essential for ⁇ v ⁇ 3 integrin dependent endothelial cell migration as well as precapillary endothelial tube formation on extracellular matrix substrates.
- ⁇ 3 integrin cytoplasmic tyrosine motif in processes of angiogenesis, wild type and DiYF mice were subcutaneously implanted with hollow fiber containing Bl 6F10 mouse melanoma tumor cells. Growth factor secreted by tumor cells rapidly induced neovascularization only in wild type mice around the hollow fibers. No such dramatic increase in neovascularization found in DiYF mice (Fig. 24 C and D), Analysis of blood vessel length also indicated significantly higher degree of neovascularization in wild type mice compared to DiYF (80 mm vs 25 mm). Thus, ⁇ 3 integrin cytoplasmic tyrosine motifs are crucial for initiation of angiogenic program in endothelial cells and ultimately regulate the processes of angiogenesis.
- ⁇ v ⁇ 3 integrin engagement by extracellular matrix proteins seems to be involved in the coordinated activation of tyrosine kinase receptors.
- DiYF knockin endothelial cells we have established that ⁇ 3 integrin cytoplasmic tyrosine motifs are crucial for integrin-dependent augmentation of VEGF signaling.
- VEGF induces an association of its receptor VEGFR-2 with ⁇ 3 subunit of ⁇ v ⁇ 3 integrin, but not with ⁇ lor ⁇ 5 integrins on endothelial cells.
- Blocking antibodies against either ⁇ v or ⁇ 3 subunit independently blocked VEGF-induced phosphorylation of ⁇ 3 integrin cytoplasmic tyrosine motifs and VEGF-induced VEGFR-2 phosphorylation.
- integrin antagonists would affect not only integrin dependent signaling pathways but would also diminish intracellular responses to VEGF.
- phosphorylation of both receptors occurred in an inter-dependent manner where phosphorylation of one component of the complex was necessary for complete activation of its partner molecule.
- c-Src as a molecule that directly phosphorylates the cytoplasmic tyrosine motifs of ⁇ 3 integrin in response to VEGF stimulation. Further, c-Src is able to directly control VEGF-induced and integrin-mediated cellular responses such as cell adhesion and migration. Src, Yes and Fyn triple mutant cells (SYF) exhibited severely impaired ⁇ 3 integrin tyrosine phosphorylation in response to growth factors and this was corrected by re-expression of c-Src alone.
- Src dependent ⁇ 3 integrin cytoplasmic tyrosine motif phosphorylation is a key step in a receptors crosstalk, since phosphorylation deficient mutant of ⁇ 3 (DiYF) did not form a complex with VEGFR-2 despite expression of constitutively active Src.
- Src might mediate a wide spectrum of growth factor-induced responses.
- Recent observations demonstrated that Src is required for VEGF-induced vascular permeability, a response triggered by VEGFR-2 activation.
- v-src is able to modulate blood vessel development in several experimental animal models, appears to be crucial for endothelial cell adhesion, motility as well as cellular response to shear stress.
- Rabbit polyclonal anti-VEGFR-2, anti- ⁇ 3-integrin, anti- ⁇ 5-integrin 5 anti- ⁇ l-integrin, anti-Src, anti-Fyn, anti-Yes, and mouse monoclonal anti-phospho- tyrosine (PY20 and PY99) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
- Anti-phospho-Src (Tyr-416), anti-phospho- VEGFR-2, and purified, recombinant Src were from Cell Signaling Technology (Beverly, MA).
- Rabbit polyclonal anti-phospho- ⁇ 3-integrin antibodies (Tyr 747 and Tyr 759) were from Biosource International, Inc. (Camarillo, CA).
- Mouse monoclonal anti- ⁇ 3integrin, anti- ⁇ 5-integrin, anti- ⁇ l-integrin, anti- ⁇ v integrin blocking antibodies were from Chemicon International, Inc. (Temecula, CA). Purified collagen, laminin, vitronectin, bFGF, and VEGF were purchased from R&D Systems (Minneapolis, MN). Matrigel was obtained from BD Biosciences
- HUVEC Human umbilical cord vain endothelial cells
- DMEM Dulbecco-modified eagle medium
- FBS fetal bovine serum
- L-glutamine L-glutamine
- antibiotics were grown at 37 0 C in 6% CO2.
- DiYF mice were generated in the laboratory of Dr. David R. Phillips and maintained on a C57/B16 background (seven generations of backcrossing). Six-to eight-week old wild-type (WT) and DiYF mice were used in this study. We performed all procedures according to protocols approved by Cleveland Clinic Foundation Institutional Animal Care and Use Committee. WT and DiYF mouse lungs were removed by surgical procedure minced and digested using collagenase- dispase reagent (3 mg/mL). Digests were strained and the resulting cell suspension was plated on flasks coated with 5 ⁇ g/mL fibronectin. The hollow fiber angiogenesis assay was performed as described previously using Bl 6F10 mouse melanoma cells. After 12 days mice were anesthetized and sites of fiber implantation were photographed and cumulative vessel lengths were calculated.
- HUVECs or mouse embryonic fibroblasts (SYF and SYF + Src) or mouse lung endothelial cells were detached from the tissue culture flasks using 20 mM EDTA. Cells were washed twice with sterile PBS and re-suspended in DMEM. The cell suspensions were added to ligand-coated wells and placed in humidified incubator for 45 min. Wells were gently washed three times with DMEM and photographs were taken. The numbers of attached and spread cells per field were counted. CeIl migration assay
- Transwell tissue culture inserts were coated with ⁇ v ⁇ 3 integrin ligand vitronectin for 24 h at 4 0 C.
- Endothelial cells were transfected with various forms of c-Src or treated with inhibitors and were trypsinized; then IXlO 5 cells were added into each well.
- Cells were allowed to migrate for 12 h and fixed with 70% methanol for 15 min and stained with Giemsa. The non-migrated cells adhered to the top surface were removed and three random 1OX fields were photographed using an inverted phase contrast microscopy.
- Endothelial cell motility on various extracellular matrix protein-coated plates induced with VEGF-Al 65 was analyzed by time-lapse videomicroscopy.
- Time-lapse imaging was performed using a Leica DM IRB microscope supported by the Metamorph program (Molecular Devices). Images were acquired every 10 min for 10 hr using a Photometric Cool Snap Camera (Roper Scientific) under 5% CO2 and at 37 0 C in a stage incubator. Cell paths were generated from centroid positions and migration parameters were computed with ImagePro plus software.
- HUVECs were transfected with various forms of c-Src.
- Cells were serum starved for 4 h and further induced with 20 ng/mL bFGF or VEGF-Al 65.
- Fluorescein isothiocyanate (FITC)-labeled fibrinogen was added at a final concentration of 200 nM and cell were incubated for 30 min.
- Cells were fixed with 3.7% formaldehyde/PBS for 15 min and washed twice with ice-cold PBS.
- Fluorescence-activated cell sorting FACS was performed using a FACS Calibur (Becton Dickinson, San Jose, CA) and data were analyzed using CellQuest software program.
- HUVECs were transfected with various forms of c-Src or Src-H-.
- SYF, SYF+Src cells were serum starved for 4 h and further stimulated with 20 ng/mL VEGF-Al 65 or b-FGF.
- WOW-I Fab was added at a final concentration of 30 ⁇ g/mL, followed by addition of FITC-conjugated goat anti- mouse IgG at 10 ⁇ g/mL. After 30 min cells were fixed with 3.7% formaldehyde/PBS for 15 min, washed twice with PBS, and FACS analysis was performed as described above.
- vascular tube-like structures by endothelial cells was assessed on a basement membrane matrix preparation. Twelve-well plates were coated with 0.5 mL of Matrigel according to the manufacturer's instructions. Wild-type and DiYF mouse lung endothelial cells were transfected with various forms of c-Src and detached from the tissue culture flasks using 20 mM EDTA. Cells were washed twice with sterile PBS and seeded on Matrigel-coated plates. Medium with or without 20 ng/mL VEGF was added and cells were further incubated at 37 0 C for 8 h. The tube formation was observed using an inverted phase contrast microscope ⁇ Leica, Wetzlar, Germany) and photographs were taken. Using ImagePro software, the degree of tube formation was quantified by measuring the length of tubes in three random fields. Immunopreeipitation and immu ⁇ oblotting
- Cells were lysed in imm ⁇ noprecipitation lysis buffer (1% Noniodet P-40, 150 mM NaCl, 50 mM Tris-HCL (pH 7.8), 2 mM EDTA, 10 mM NaF, 10 mM Na2P2O7, 2 mM Na3VO4 10 ⁇ g/mL leupeptin, 4 ⁇ g/mL pepstatin and 0.1 U/mL aprotinin). Cell lysates were centrifuged at 13,000xg for 10 min at 40 0 C. Supernatants were collected and assayed for protein concentration using the Bio- Rad protein assay method.
- imm ⁇ noprecipitation lysis buffer 1% Noniodet P-40, 150 mM NaCl, 50 mM Tris-HCL (pH 7.8), 2 mM EDTA, 10 mM NaF, 10 mM Na2P2O7, 2 mM Na3VO4 10
- Cell lysates containing 700-800 ⁇ g of total protein were pre-cleared and were immunoprecipitated with the indicated antibody. Immunocomplexes were denatured using Laemmli sample buffer and proteins were separated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and probed with indicated antibody. When appropriate, nitrocellulose membranes were stripped and blotted according to manufacturer's instructions. Endothelial cell surface biotinylation and Western blot analysis were performed.
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Abstract
Méthode de modulation de l'angiogenèse dans un tissu, consistant à administrer audit tissu une dose efficace au plan thérapeutique d'un agent qui module la formation complexe d'αvβ3 intégrine et de VEGFR2.
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WO2009043104A1 (fr) * | 2007-10-04 | 2009-04-09 | Bionomics Limited | Marqueurs de cellules endothéliales et leurs utilisations |
US20140228295A1 (en) * | 2010-03-15 | 2014-08-14 | The Board Of Trustees Of The University Of Illinois | Inhibitors of Beta Integrin-G Protein Alpha Subunit Binding Interactions |
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AU4815397A (en) * | 1996-10-15 | 1998-05-11 | Vanderbilt University | Method of disrupting cellular adhesion |
JP2001506595A (ja) * | 1996-11-21 | 2001-05-22 | シーオーアール セラピューティクス,インコーポレイテッド | ミオシンとインテグリンとの間の相互作用の調節 |
WO2001079144A2 (fr) * | 2000-04-14 | 2001-10-25 | Cor Therapeutics, Inc. | Fyn kinase comme cible de modulation de la transduction des signaux medies par l'integrine |
CA2410509A1 (fr) * | 2000-06-02 | 2001-12-13 | Sugen, Inc. | Derives d'indolinone comme inhibiteurs de phosphatase/proteine kinase |
US6958340B2 (en) * | 2001-08-01 | 2005-10-25 | Merck & Co., Inc. | Tyrosine kinase inhibitors |
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US3773919A (en) | 1969-10-23 | 1973-11-20 | Du Pont | Polylactide-drug mixtures |
US4244946A (en) | 1979-06-11 | 1981-01-13 | The Salk Institute For Biological Studies | Water-soluble peptides affecting gonadal function |
US5712291A (en) | 1993-03-01 | 1998-01-27 | The Children's Medical Center Corporation | Methods and compositions for inhibition of angiogenesis |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2009043104A1 (fr) * | 2007-10-04 | 2009-04-09 | Bionomics Limited | Marqueurs de cellules endothéliales et leurs utilisations |
US8309351B2 (en) | 2007-10-04 | 2012-11-13 | Bionomics Limited | Methods of identifying agents that inhibit angiogenesis |
AU2008307145B2 (en) * | 2007-10-04 | 2014-07-10 | Bionomics Limited | Markers of endothelial cells and uses thereof |
US20140228295A1 (en) * | 2010-03-15 | 2014-08-14 | The Board Of Trustees Of The University Of Illinois | Inhibitors of Beta Integrin-G Protein Alpha Subunit Binding Interactions |
US9156884B2 (en) * | 2010-03-15 | 2015-10-13 | The Board Of Trustees Of The University Of Illinois | Inhibitors of beta integrin-G protein alpha subunit binding interactions |
US10011634B2 (en) | 2010-03-15 | 2018-07-03 | The Board Of Trustees Of The University Of Illinois | Inhibitors of beta integrin-G protein alpha subunit binding interactions |
US10738080B2 (en) | 2010-03-15 | 2020-08-11 | The Board Of Trustees Of The University Of Illinois | Inhibitors of beta integrin-G protein alpha subunit binding interactions |
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WO2008011513B1 (fr) | 2008-10-16 |
EP2068877A4 (fr) | 2011-09-21 |
WO2008011513A3 (fr) | 2008-08-14 |
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